GIFT OF
WASH. ACADEMY" OF SCIENCES
[ UNIVERSITY
V OF
X£
WASHINGTON ACADEMY OF SCIENCES
VOL. VI, PP. 1-202. SEPT. 10, 1904,
CONTRIBUTIONS TO THE KNOWLEDGE OF THE
LIFE HISTORY OF PINUS WITH SPECIAL
REFERENCE TO SPOROGENESIS, THE
DEVELOPMENT OF THE GAMETO-
PHYTES AND FERTILIZATION.
BY MARGARET C. FERGUSON, PH.D.,
ASSOCIATE PROFESSOR OF BOTANY, WELLESLEY COLLEGE.
PLATES I-XKIV.
CONTENTS.
Introduction.
Purpose of the study 3
Historical notes 5
Methods.
Collecting 12
Fixing 13
Staining 15
CHAPTER I. Microsporogenesis.
The microsporangium.
The wall of the pollen-sac 17
The primitive archesporium 18
Tetrad-division.
The definitive archesporium 20
The first nuclear division of the microspore-mother-cell 21
The second mitosis of the mother-cell 30
The problem of reduction 31
Development of the microspore.
The formation of the spore-wall 34
The origin of the air-sacs and liberation of the microspore .... 36
The growth of the microspore 37
Summary 39
Proc. Wash. Acad. Sci., September, 1904. (i)
2 MARGARET C. FERGUSON
CHAPTER II. The male gametophyte.
The development of the pollen-grain.
The formation of the prothallial cells 41
The mature pollen-grain 44
Pollination.
The ovule at the time of pollination 45
The pollen-chamber 46
Development of the pollen-tube.
The first period of growth.
Germination of the pollen-grain 47
The division of the antheridial cell 48
The winter condition 50
The second period of growth.
Renewed activities in the macrosporangium 50
Renewed activities in the male gametophyte 51
Division of the generative nucleus 53
Growth of the sperm-nuclei 62
Elongation of the pollen-tube 64
Summary 66
CHAPTER III. Macrosporogenesis.
The female cone.
The macrosporangium 70
Formation of the axial row.
The macrospore-mother-cell 71
The first division of the mother-cell 72
The second division of the mother-cell 74
Significance of the tetrad-division within the ovule 76
Later history of the axial row.
The fate of the upper cells 79
The growth of the macrospore So
Summary 81
CHAPTER IV. The female gametophyte.
Development of the prothallium.
The first period of growth 82
The second period of growth 84
The so-called " spongy tissue."
The first period of growth 86
The second period of growth 88
The nature and function of this tissue 89
Development of the archegonium.
The early growth of the archegonium 90
The division of the. central cell 95
History of the ventral canal-cell 97
Maturation of the egg.
The descent and growth of the egg-nucleus 99
The " proteid vacuoles " 103
The receptive vacuole 109
Summary no
LIFE HISTORY OF PINUS 3
CHAPTER V. Fertilization and related phenomena.
Conjugation.
The coming together of the gametophytes 113
The union of the sexual nuclei 114
The first division following fecundation.
The prophases of the division 115
Later stages in the mitosis 118
The pro-embryo.
Division of the two segmentation-nuclei 122
The four segmentation-nuclei 124
The development of cell-walls 126
Later mitoses in the formation of the pro-embryo 127
The fate within the egg of the smaller sperm-nucleus, the stalk-
cell, and the tube-nucleus 128
Summary 130
APPENDIX. Some abnormal conditions.
Supernumerary nuclei in the male gametophyte 133
Unusual conditions in the female gametophyte 135
A peculiar method of conjugation 138
NOTE 139
LIST OF PAPERS CITED 142
EXPLANATION OF PLATES 154
INTRODUCTION.
THERE is no chapter in the annals of botanical science
more fascinating than that which deals with the history of
sexuality in plants. No definite date marks the discovery of
the fact that plants, like animals, are male and female ; the
idea was rather a growth, as is plainly shown by the writings of
Aristotle, Theophrastus, Pliny and others of the early philoso-
phers. The fact may, however, be said to have been estab-
lished by Camerarius (1694) in his " De sexu Plantarum," but
for many years after his time botanists found in this question
merely a favorite subject for philosophical speculation. Their
ideas remained vague and uncertain, no effort being made to
confirm their theories either by observation or experimentation.
It was not until near the middle of the last century that actual
investigations were begun along this line. Amici (1830-1846)
made certain interesting observations regarding the develop-
ment of the pollen-tube and the origin of the embryo in several
plants ; but the splendid series of embryological papers pub-
lished by Hofmeister (1848-1867) first placed the science upon a
sure foundation and marked a new era in the study of sexual
4 MARGARET C. FERGUSON
reproduction in plants. Although the researches of Hofmeis-
ter, Strasburger, Warming, Belajeff and others who have con-
tributed to our knowledge of this subject, especially during the
last decade, have disclosed many facts concerning the structure
and development of the pollen-grain, of the ovule and of the
embryo, our knowledge of certain phases of spermatogenesis
and oogenesis is still very meager, and not a sufficiently large
number of plants have been thoroughly investigated to admit of
generalizations. The celebrated discoveries of Hirase, Ikeno
and Webber, in 1897, gave a new incentive to this study, par-
ticularly in connection with the Gymnosperms, and rendered it
highly desirable that fertilization and associated phenomena
should be worked out for other members of this group by the
more modern methods of investigation.
The present studies were begun in the fall of 1897 with
the hope of adding somewhat to our knowledge of this subject.
Incidentally, it seemed desirable to determine whether any ves-
tiges of the bodies called blepharoplasts by Webber (i8973) still
persist in the conifers. As a result of the past embryological
studies, a vast number of facts pertaining to the life-history of
the gametophytes in the higher platns has accumulated. While
many of the conclusions reached are the outcome of serious
direct investigations, others are based on the insufficient evi-
dence found in a rather superficial study of a large number of
plants. What we need to-day is not more facts regarding un-
related plants, so much as a careful working out of .the details
of development in representative genera.
This research is based primarily upon a study of Pinus
Strobus, but nearly every observation recorded has been con-
firmed for Pinus rigida and P. austriaca^ and to a large extent,
for P. montana var. uncinata and P. resmosa. The descrip-
tions given may be understood to refer alike to the five species
named above unless otherwise stated in the text. Nearly six
hundred paraffin blocks with imbedded material have been
made, and more than four thousand slides of serial sections
have been stained and studied. Six hundred separate collec-
tions of material would seem unnecessarily large if one were
studying a plant like Nicotiana in which, according to Guig-
LIFE HISTORY OF PINUS 5
nard (1902), fertilization follows in 2 hours after pollination, but
in Pinus i where almost 13 months intervene between these two
processes, such a number is not excessive. While it is true in
cytological studies, as elsewhere, that numbers, or mere mass
work, do not signify excellence, it is equally true that the re-
sults of investigations based upon a study of a limited amount
of material are, at best, unsatisfactory, and, other things being
equal, those conclusions will be most valuable which have been
formulated after a careful observation of many specimens.1
HISTORICAL NOTES.
In the following brief summary of the literature dealing
with the Abietincce, only the more important papers have been
noted, and the observations recorded by the various writers
have been given without comment.
The tetrad-division in the pollen-mother-cell of Pinus and
Abies was studied in 1848 by Hofmeister. He stated that the
pollen-mother-cells were already developed in the anthers at the
end of November, two special daughter-cells were formed at
the close of the first division in the spring, and the four cells
resulting from the second division were found to lie either in
one plane or at the corners of a tetrad. Three years later
(1851) Hofmeister published the results of his remarkable series
of investigations in the higher cryptogams and conifers. He
described and figured the pollen-grain in the Abietinece as con-
sisting of a cell-complex, noted the depression in the apex of
the nucellus in Pinus at the time of pollination, and the single
embryo-sac-mother-cell deep in the interior of the nucellus. It
appeared that the pollen-grain rested some weeks upon the
nucellus before the pollen-tube was emitted. After the germina-
tion of the pollen-grain, the tube grew for several weeks and
penetrated nearly to the point of union between integument and
nucellus, but it might cease growth before so great a depth was
reached.
1 This paper was given especial honorable mention on April 26, 1903, by the
Association for Maintaining the American Women's Table at the Zoological
Station at Naples and for Promoting Scientific Research by Women. I wish here
to express my deep gratitude to Mrs. Ellen H. Richards, Miss Florence Gushing
and other members of the above named association through whose generous
efforts the publication of this paper in its present form has been made possible.
O MARGARET C. FERGUSON
He concluded that the embryo-sac remained for a long time
as a single cell, its nucleus finally dissolving to be replaced
by a number of free nuclei ; in a few days the sac was filled
with long cells reaching to the middle ; at the beginning of
winter, the walls of this transitory endosperm were greatly
thickened ; in the spring, the thickened walls of the endosperm
were absorbed and the cells liberated. Each primordial cell
thus made free contained, somewhat later, three or four
daughter-cells which were, in their turn, liberated by the disso-
lution of the mother-wall. Thus the number of cells within
the embryo-sac was greatly increased, the embryo-sac itself
growing to more than twenty times its previous volume. The
cells of the nucellus also multiplied rapidly except in the region
previously penetrated by the pollen-tubes. In the middle of
May, a layer of cells lined the embryo-sac, cell layers in-
creased until they met in the center, then the corpuscula were
differentiated. The corpuscula were always separated in the
Abietinece by one or more layers of cells, and the walls enclos-
ing the corpuscula were thought to be channelled, thus afford-
ing open communication with the surrounding cells. In Pinus
from 3 to 5 corpuscula were developed in each ovule, and a
corresponding number of funnel-shaped openings occurred in
the upper part of the endosperm. When the pollen-tube reached
the corpusculum it contained free spherical cells in its lower
end. The tube either flattened itself out upon the corpusculum
or penetrated a short distance into it. After fertilization the
impregnated germinal vesicle increased in size, its nucleus dis-
appeared, and soon a large daughter-cell was seen at the base
of the corpusculum. By repeated divisions of this cell the pro-
embryo was formed.
In 1858 Hofmeister found the usual number of neck-cells in
Pinus Strobus to be four, exceptionally three, five, or six, all
lying in the same plane. He further demonstrated the vacu-
olate character of the contents of the corpusculum during its
development. These vacuoles disappeared before impregna-
tion, being replaced by free cells — the germinal vesicles, or
Keimblaschen. A pit was figured in the apex of the pollen-
tube after its entrance into the corpusculum, but it was said that
LIFE HISTORY OF PINUS 7
the tube remained closed until after the formation of the pro-
embryo, when it was ruptured by mechanical means. The
great abundance of starch in the pollen-tube of the Abietinece
was also mentioned at this time. While the " Higher Crypto-
gamia " appearing in 1862 was largely a translation of Hof-
meister's earlier publications, it likewise presented many new
observations. The fact was noted that in Pinus the integument
surrounds the nucellus, leaving open above its apex a wide
micropylar canal. In all the Conifer a > after the embryo-sac
was entirely filled with cellular tissue, certain cells near the
micropylar end ceased dividing but increased markedly in size ;
the other cells of the endosperm continued to multiply in num-
ber, but remained comparatively small ; thus the corpuscula
were differentiated. After the cutting off of the neck-cells in
the Abietinece , additional cells were developed at the top of the
endosperm, giving rise to the depressions referred to in 1851.
Scarcely a day intervened between the approach of the pollen-
tube and the formation of a four-celled pro-embryo at the base
of the corpusculum, and this occurred contemporaneously in all
ovules of all trees growing under similar circumstances.
The works of Strasburger on this subject have been more
numerous and complete than those of any other investigator. It
is extremely interesting to note how his interpretations have kept
pace with the improvements in methods of research. In 1869
he traced the development of the endosperm from the free cells
lining the embryo-sac to its maturity, and established the fact
that shortly before fertilization the central cell divides to form
the canal-cell and the egg-cell. He confirmed Hofmeister's
observations regarding the channels in the upper part of the
endosperm, and the presence of a closed pit at the apex of the
pollen-tube ; but he did not observe the nuclei in the pollen-tube,
and remarked that, inasmuch as the sexual organs touch "in
these plants, spermatozoids would be superfluous and were, in
reality, not present. He added, however, that their place was
taken by granular protoplasm and starch grains which exercised
the same fertilizing effect on the egg as do spermatozoids.
After fertilization four nuclei were detected at the base of the
corpusculum and a division into a cross took place, these cells
MARGARET C. FERGUSON
divided and were separated by cross-walls, the lower four di-
vided again making three layers of four cells each, the middle
layer then elongated pushing the lowest cells down into the
endosperm. In Picea a fourth layer of cells was observed at
the base of the central cell.
In 1872 Strasburger stated that the canal-cell loosened itself
from the egg and hung as a cap just beneath the neck-
cells, at the same time the egg-nucleus increased in size and
moved to the center of the corpusculum. He detected two cells
in the pollen-tube of several Gymnosperms, but considered that
such cells were extremely rare in the Abietinece^ as he had only
once found one in this group. The shrunken remains of these
cells were seen in the pollen-tube after fertilization. He be-
lieved that the pit of the pollen-tube remained closed, and that
the exchange-substance was apparently communicated by a
vacuole between the apex of the pollen-tube and the egg-
nucleus. After fertilization the central nucleus was dissolved,
and, in " abnormal " cases, four new nuclei appeared in the
central part of the egg, but there was strong evidence that
these did not develop into an embryo. Six years later (1878),
he observed one or more divisions in the pollen-grain shortly
before pollination. The small cells resulting from these divi-
sions were interpreted as rudimentary prothallium. Two large
primordial cells were demonstrated in the pollen-tube of Pinus
and Picea when the tube was just above the archegonium. Ac-
cording to Strasburger's interpretation at that time, the nucleus
in front was dissolved while the one behind entered the egg
and fused with its nucleus. This was a great advance on his
previous observations, but he still conceived of the pollen-tube
as remaining closed, and fancied that the protoplasmic contents
passed through the membrane directly while the starch was dis-
solved before its transmission into the egg. He was now con-
vinced that only a part of the contents of the pollen-tube was
taken up by the egg-nucleus, the remaining portion uniting di-
rectly with the egg-plasma ; but he was not certain whether the
protoplasm active in fertilization came in as a formless mass or
in the shape of a nucleus.
Strasburger established the fact, in 1879, tna* ^ *s tne
LIFE HISTORY OF PINUS
9
most of the two sperm-nuclei in the pollen-tube which is instru-
mental in effecting fertilization. He reported the presence of
an axial row of three cells in Larix, the lowest cell being the
embryo-sac-mother-cell. The generalization was made that
the prothallium arises in all the gymnosperms through free
cell-division, all the free nuclei dividing at the same time. It
was claimed that but a single endosperm was formed in the
Abietinea, that the primary nucleus of the embryo-sac remained
undivided during the first year, and that the " transitory endo-
sperm " of Hofmeister was in reality the freed cells of the nucel-
lus which were destined to be absorbed. It was to these cells
that the term spongy tissue was applied. In the following year
(1880) Strasburger described and figured the mature archego-
nium in Picea and discussed the early stages of endosperm for-
mation in Pinus, but he gave little that was new at that time.
It was in this same year that Sokolowa (1880) published the
results of her researches in the development of the prothallium
in the gymnosperms. Cell-walls were laid down between the
nuclei imbedded in the peripheral layer of protoplasm, but no
cell thus formed was furnished with a wall on its inner free side.
These open cells were termed " alveoli." They grew in length
until the middle of the embryo-sac was reached, then walls
arose at the inner ends and the alveoli were closed ; cell divis-
ions followed, and gradually the elongated alveoli gave place
to many cells.
Goroschankin (1880 and '83) reported that the protoplasm of
the egg and of the sheath-cells was in immediate contact through
pores in the separating membrane ; he saw (i8832) the two
sperm-nuclei pass into the egg in Pinus Pumilio^ and believed
that both fused with its nucleus ; the great similarity which
the spheres in the egg bear to nuclei was commented upon and
he questioned the propriety of calling them vacuoles. Stras-
burger (1884) confirmed Goroschankin's observations as to the
passage of the two sperm-nuclei from the pollen-tube into the
egg, but pointed out that only the one in advance fused with the
egg-nucleus. As the protoplasmic contents of the central cell
increased, the vacuoles decreased, and every transition could be
traced between the large vacuoles and the meshes of the proto-
Proc. Wash. Acad. Sci., July, 1904.
OF
10 MARGARET C. FERGUSON
plasm filled with metaplasm. In the pines, a large vacuole
often held several smaller ones. The egg-nucleus slowly filled
itself with metaplasm during its descent to the center of the
cell. Three successive divisions occurred in the large cell of
the pollen-grain in Lartx, the first two prothallial cells formed
were small and soon disorganized, the third one increased greatly
in size and divided to form the stalk- and the body-cell.
It was left for Belajeff (1891) to establish the true nature of
the cell-complex found in the pollen-grain of the Gymnosperms.
He demonstrated the fact that in Taxus baccata the large nucleus
of the pollen-grain is the vegetative or pollen-tube-nucleus, as
in the Angiosperms, and that the sperm-nuclei arise by the
division of one of the smaller cells of the pollen-grain, this
smaller cell first dividing to form the stalk- and the generative
cell.
Strasburger (1892) showed that Belajeff 's observations on the
structure of the pollen-grain and the development of the pollen-
tube in Taxus baccata were, in general, true for the other
Gymnosperms. He described the mature pollen-grain in Pinus
as containing a large tube-cell, a small cell — the third prothallial
or antheridial cell — and the remnants of the first two prothallial
cells. Pollination was immediately followed by the germination
of the pollen-grain, and the nucleus of the large cell wandered at
once into the tube. The last formed prothallial cell remained in
its place in the pollen-grain until the following spring, when it
divided into the stalk- and the body-cell of the antheridium.
The division of this cell was not studied, but Strasburger
thought it took place at about the same time as the develop-
ment of the archegonia. The pollen-grain of Picea was found
to correspond exactly with that of Pinus excepting that the an-
theridial cell divided while still within the anther. The sperm-
cells in Pinus were seen in the apex of the pollen-tube ; the
lower cell was the larger ; and each cell was almost entirely
filled with its large, coarsely granular nucleus. At the tip of
the pollen-tube, the stalk- and the tube-nucleus could no longer
be distinguished one from the other. The sperm-nucleus was
shown to be smaller than the egg-nucleus, but the two were
alike in the amount of active nuclear substance ; and attention
LIFE HISTORY OF PINUS II
was called to the smallness of the first nuclear figure following
fecundation in comparison with the size of the conjugating
nuclei. The germ-nucleus divided in its original position in the
egg, and the two nuclei passed towards the " organic" apex of
the archegonium.
Belajeff (1893) worked out the development of the pollen
tube in Picea as a type of the Abietmecz. He found that the
generative cell divided while still within the pollen-grain and
gave rise to two sperm-cells which he figured as of the same size.
Dixon (1894) traced the history of the pollen-grain and the
pollen-tube in Pinus sylvestris from the middle of April to the
time of fertilization. He thought that the prothallial cell divided
towards the end of April to form a small stalk-cell and a larger
body-cell. The body-cell immediately divided into two cells of
almost equal size — the male sexual cells. The sperm-cells
moved into the pollen-tube followed by the nucleus of the stalk-
cell. Pollen-tubes were found to branch freely while in the
upper " brown" tissue of the nucellus but only one branch of
each tube was continued through the lower part of the nucellus.
He noted that the four nuclei, much of the protoplasm, and
considerable of the starch of the pollen-tube passed into the
oosphere. As a rule, eight chromosomes were found in the
nuclei of the female gametophyte.
In giving an account of some work done by his students on
the Gymnosperms, Coulter (1897) reported that the work of
Dixon " was largely confirmed in the minutest detail"; and in
1900 he figured the pollen-tube "in pines," when just above
the archegonium, showing two sperm-cells of equal size. Atkin-
son (1898) stated that the sperm-mother-cell in Pinus divided
into two sperm-cells after having passed into the pollen-tube.
Blackman's excellent treatise on fertilization and related
phenomena in Pinus sylvestris was published in 1898. Many
details of development were most carefully worked out, but the
facts recorded are not enumerated here, since they will be duly
considered in connection with the observations, as record ed
in the body of this paper, that have been made by the writer on
other species of pines. Since the appearance of Blackman's
monograph, a considerable literature dealing with various stages
12 MARGARET C. FERGUSON
of development in the gametophytes of the Abietinece. has been
published. The details of these investigations are familiar to all
students of the subject. These papers will, therefore, be men-
tioned at this point by title only ; they will be referred to again
in the discussions which follow. Chamberlain (1899), Oogene-
sis in Pinus Laricio ; Wuicizki (1899), Ueber die Befruchtung
bei den Coniferen ; Arnoldi (1900), Beitrage zur Morphologic
der Gymnospermen, IV; Juel (1900), Beitrage zur Kenntniss
der Tetradentheilung ; Murrill (1900), The Development of
the Archegonium and Fertilization in the Hemlock Spruce
(Tsuga canadensis Carr.); Coulter and Chamberlain (1901),
Morphology of the Spermatophytes ; Ishikawa (1901), Reduc-
tion Division in Larix ; and the papers published by the writer
in 1901. l
METHODS.
Collecting. — On November 15, 1897, and each week there-
after until December 25, cones of Pinus Strobus, P. rigida, P.
austriaca, P. montana var. uncinata, and the staminate strobili
of P. austriaca were collected. Material was brought in occas-
sionally during the remainder of the winter. Pistillate cones
of the species named, and also of P. resinosa, were collected
once each week beginning with April i ; collections were made
twice each week throughout the month of May, and three times
a week during June. From June 10-30, a period which was
sure to cover fertilization, cones of Pinus Strobus were collected
every day at about nine o'clock in the morning, and frequently
again at four o'clock in the afternoon. Male cones were gath-
ered, from those species in which they had appeared, at irregu-
lar intervals during the early spring. From the first of May
until the time of pollination, which varies by a number of days
in the different species, staminate strobili were collected each
day. During May and June the young female cones were
gathered as well as the more mature ones of the previous year's
growth. After July i, the older cones were no longer collected,
but the young cones of Pinus Slrobus, P. rigida, and P. austri-
aca were collected once each week until November 15. Cones
1 See " Note " at close of Appendix.
LIFE HISTORY OF PINUS 13
of Pinus Strobus were again collected regularly, as described
above, throughout the spring and early summer of 1899. Collec-
tions of the staminate cones of Pinus Strobus and P. rigida
were made during May and June 1901, and from May 15 to
June 15 of the same year the young pistillate cones of Pinus
rigida were gathered daily.
Material was obtained from different trees and different locali-
ties. The practice of collecting all one's material from a single
tree, as reported by Murrill (1900), Land (1902) and others, does
not seem a safe one to follow, for certain peculiarities of develop-
ment which are not characteristic of the species may appear in
a*n individual. At the time of each collection, ovules were put
up from several cones of each species, these cones being taken
not from the tip of one branch but from different branches.
The central portion only of the cone was used, the ovules at
either extremity being more or less abortive. After collecting,
the material was taken at once to the laboratory and preserved.
The staminate cones and, in the early stages of development,
the pistillate ones were fixed entire or cut into quarters longitu-
dinally. Very soon the individual scales of the female cones
were removed from the receptacle before fixing, and, when
the scales were of sufficient size to admit of such manipu-
lation, all superfluous parts were cut away, leaving the two
tiny ovules still united by a small portion of the scale.
With the renewal of growth in the spring, the ovules were
removed from the scales and, as soon as it was feasible,
a portion of the integument was cut away from two or
more sides of each ovule, thus bringing the fixing fluid into
direct contact with the young gametophyte. For later stages,
the endosperm was frequently removed from the integument,
but such material did not prove to be as satisfactory as that in
which the nucellar cap and a small portion of the coat were
left in connection with the prothallium. Throughout the entire
mechanical process of preparing material for the fixer, the most
extreme care was used, as it was found that a very slight pres-
sure was sufficient to cause distortions and thus to render the
material worthless for cytological studies.
Fixing. — The methods used in fixing and staining do not
14 MARGARET C. FERGUSON
differ materially from those generally employed in cytological
work. The fixing fluids tested were chrome-osomo-acetic acid
solution, chrome-acetic acid solution, corrosive sublimate in
aqueous solution, absolute alcohol, and Carnoy's fluid. The
first two were tried with variations in concentration and in length
of time. The chrome-osomo-acetic acid solution giving by far
the best results, the other fixers were entirely discarded. It
was made up according to the following formula :
Chromic acid crystals 1.3 gms.
Osmic acid (in glass bulb) 5 gms.
Glacial acetic acid 83 c.c.
Distilled water 160.0 c.c.
ii
This solution used in one half strength and allowed to act for
about 15 hours proved to be most excellent for fixing the pro-
thallium at the time when it consists of a wall layer of proto-
plasm containing numerous free nuclei. For the development
of the pollen-grain and fertilization stages, it was most satisfac-
tory when undiluted, and allowed to act for about 24 hours. If
the fluid blackened at all, it was poured off after 2 or more
hours and fresh added.
After fixing, the material was washed in running water from
2 to 12 hours, but as a rule specimens were not kept in the
running water longer than 6 hours. The very convenient piece
of apparatus described by Durand ('99) was used for this process.
Subsequent to washing, material was dehydrated in 8 grades of
alcohol beginning with 15^ and ending with the absolute. It
was not allowed to stand in the lower grades for more than 6
hours, and was rarely kept in the absolute alcohol longer than
that time ; the latter was changed 3 times, once about every 2
hours, to insure perfect dehydration in as short a time as pos-
sible. After material had been in 85^ alcohol for 12 hours, it
was decolorized in a 35^> solution of hydrogen peroxide, made
up in 95^ alcohol, for 24 hours. While material was always
bleached in toto, it was frequently found necessary to decolor-
ize again on the slide. After dehydration, material was brought
gradually, through ascending grades, into pure cedar oil, xylol
or chloroform. The best results were obtained with the cedar
oil and it was far more commonly used than the others. If it
LIFE HISTORY OF PINUS 15
was desirable to store material for a few days or weeks, pure
cedar oil was found to be a much better medium than 75 ft
alcohol, which is commonly used for temporary storing of ma-
terial. For the purpose of getting specimens into pure paraffin
they were transferred to tiny wire-gauze baskets and carried
successively into 25, 50 and 75$6 paraffin in cedar oil, and
finally into pure paraffin with a melting point of "54°, in which
they were at last imbedded. This is a very convenient and eco-
nomical method for getting material through the paraffin oven.
The grades of cedar oil in paraffin can be kept in the bath a
long time and used repeatedly with impunity, and material can
be carried in the little baskets from bottle to bottle much more
quickly and with less liability to injury than in any other way
with which I am familiar. At the time of fixing, a small piece
of paper, bearing the number, in pencil, corresponding to the
number of the entry in the record book, was placed in each
bottle, remained with the material through all the changes which
followed, and was finally imbedded in one corner of the paraffin
block containing the specimens.
Staining. — A Minot-Zimmermann revolving microtome was
used in cutting the material. The sections varied in thickness
from 4 to 13.6 microns, but by far the greater number were
made 6.3 microns thick. They were fastened to the slide by
means of albumen-fixative, and the slides were labelled with
glass-ink. In preparing this ink, a paste was made of the best
English vermilion in sodium silicate, and sufficient water was
added to give the proper consistency for writing. Glass-pen-
cils, Higgins' waterproof ink, both with and without collodion,
and other methods for marking slides were tried ; but I have
never found anything at all comparable, for excellence, with
the glass-ink. When properly prepared it is not dissolved dur-
ing the process of staining, but can be removed from slides or
dishes, when desirable to do so, by heating in a strong solution
of potash or in gold dust.
As is usual in cytological studies, considerable experimenta-
tion was necessary before satisfactory stains were obtained.
Among the stains tested were Rosen's ('92) fuchsin and methy-
lene-blue method ; the Ehrlich-Biondi-Heidenhain mixture, as
1 6 MARGARET C. FERGUSON
prepared by Dr. G. Griibler ; Guignard's combination of methyl
green, acid fuchsin, and orange G; Flemming's safranin-
gentian-violet-orange combination : and Heidenhain's iron-
haematoxylin. The last two proved the most satisfactory and
were almost exclusively used. The iron-haematoxylin was fol-
lowed by orange G, or, if it was desirable to stain cell-walls,
by Bismarck brown. Iron-haematoxylin followed by Flem-
ming's triple stain, or by gentian-violet and orange G, brought
out the so-called kinoplasmic structures with great definiteness.
The best differentiation was obtained with the iron-haematoxy-
lin by allowing the haematoxylin to act from 12 to 18 hours,
decolorizing in iron-alum, and then washing in running tap-
water from 2 to 6 hours. Flemming's triple stain was often
used without the safranin with excellent results. Both anilin
and aqueous solutions of gentian-violet were used. As a rule,
a one-half percent, solution was employed, the slides remain-
ing in it from 5 to 20 minutes. The achromatic figures in the
divisions of the pollen-mother-cell, especially in Pinus Strobus,
were, however, brought out with great difficulty with this stain.
The best results were obtained for these stages by allowing the
slides to stand from 24 to 48 hours in stender dishes of distilled
water to which not more than 10 drops of a one percent, solution
of gentian-violet had been added. Pinus sections take the
orange with such avidity, that a fraction of a minute was in all
cases a sufficiently long time to allow this stain to act. After
washing out the superfluous gentian-violet and' dehydrating in
absolute alcohol, differentiation was effected by dashing with
clove oil. Bergamot oil was used for fixing and clearing, and
I have found it expedient to pass the slides from bergamot oil
to jars of xylol. They can remain in the xylol for hours, if
desirable, without injury, and the xylol is so readily miscible
with the balsam that the preparations become clear and more
satisfactory for studying in a much shorter time than when car-
ried directly to the balsam from the bergamot oil.
LIFE HISTORY OF PlNUS 1 7
CHAPTER I.
MlCROSPOROGENESIS.
THE MICROSPORANGIUM^
The Wall of the Pollen-sac. — With the exception of Pinus
Strobus, the staminate cones, in the pines which I have studied,
make their appearance in October or November. I have
searched repeatedly in the autumn for the male inflorescences
of Pinus Strobus but have never been able to find them until
late April or early May of the following spring. If they are
present at all before spring they can be scarcely more than
potentially so, for they are not sufficiently developed to be
detected in the field, nor by careful dissection in the laboratory.
/~The structure of the microsporangium agrees perfectly with
that usually described for the Abietinece. The wall of the
young pollen-sac consists of three or four layers of cells. The
cells of the outer layer are nearly isodiametric, while those of
the inner layers are smaller and more or less tabular in outline.
Just within, and in immediate contact with the archesporium, is
the ring of tapetal cells. In the early stages of development
the wall-cells are rich in cytoplasm and contain nuclei
which are large in proportion to the size of the cells. The
microsporangium increases much in size in the spring, and by
the time that the microspore-mother-cells are in the prophase of
division, considerable change has occurred in the wall-cells of
the pollen-sac. The outer layer has lost its nuclei and the cells
have become filled with a homogeneously staining resinous sub-
stance ; in Pinus Strobus this resinous deposit extends to the
second layer of wall-cells as well ; the cells of the inner layers
have been considerably flattened out, and their cytoplasmic con-
tent has become much reduced. When the pollen-grains are
mature, all the wall-cells of the microsporangium, except the
outermost layer, have disappeared. They have doubtless been
absorbed, their substance contributing to the nutrition of the
pollen-grains.
The tapetum cannot be distinguished during the earlier stages
of development from the other tissues. It is first clearly differ-
1 8 MARGARET C. FERGUSON
entiated in the spring, when the mother-cells are in the early
prophase of the heterotypic division. The mitoses leading to
development of this layer have not been studied, but there are
indications that it is formed from the outer layer of the sporog-
enous tissue rather than, as usually described, from the inner
layer of wall-cells. The microsporangium-wall, after the
appearance of the tapetum, is composed, as before, of three
or four layers of cells ; furthermore, the tapetum is always inti-
mately associated with the sporogenous tissue, while it is fre
quently found separated from the wall of the pollen-sac, probably
as a result of imperfect fixation. The question as to the origin
of this tissue in Pinus must, however, await further investiga-
tion. During the later stages of division in the pollen-mother-
cells, the tapetal cells increase much in size, their cytoplasm
becomes very dense and each cell comes to have from one to
three nuclei which have been observed in all stages of fusion.
Karyokinetic figures have been frequently noted in the tapetal
cells indicating that the nuclei of these cells divide mitotically,
and the division conforms to the ordinary or typical method of
mitosis. When the young microspores become free, these cells
have attained to their greatest size, and show a diffuse reaction
to stains. From this time they gradually diminish in size and
finally disappear altogether. The nutritive function of this
tissue is too well understood to require discussion here.
The Primitive Archesporium. — With the exception of Pinus
Strobus, the primitive archesporium is clearly differentiated
in the autumn, but the mother-cells of the microspore do not
arise until the latter part of April, and in Pinus Strobtis not until
about three weeks later.
In the younger stages of development, a superficial study
shows no sharp demarcation between archesporium and wall,
but a careful examination reveals certain differences by which
the two can always be distinguished. The cells of the arche-
sporium are larger, have larger nuclei, and denser cytoplasm
than those of the wall. They are also polyhedral in outline
while the wall-cells are somewhat tabular from the first, though
not so markedly so as at a later period. During the winter, the
nucleus of a primitive archesporal cell contains several nucleo-
LIFE HISTORY OF PINUS IQ
lus-like bodies, of which as many as eleven have been counted
in a single section of a nucleus, and a less number than seven
is rarely found. The delicate but extensive nuclear reticulum
is slightly chromatic and stains scarcely more strongly than the
cytoplasm of the cell. 7 Both cytoplasm and nuclear network
stain diffusely with gentian-violet during this period of rest
(fig. i)-
In those species in which the microsporangia make their
appearance in the autumn, the pollen-sacs remain small and
the archesporial cells comparatively few in number until the
following spring. Hofmeister ('48) found the mother-cells of
the pollen-grains in the anthers of Pinus and Abies at the end
of November, Belajeff ('94) observed the pollen-mother-cells of
Larix in the spireme stage in October, and Coulter and Cham-
berlain ('01) have recently figured the * < microsporangium of Pinus
Laricio in the mother-cell stage in October." The sporogenous
tissue, as they have illustrated it, bears a very strong resem-
blance to that shown in fig. i of this paper. There is undoubted
evidence that these are not pollen-mother-cells in the species of
pines which I have studied. In the first place, the number of
cells in a single anther in November is far less than the number
of microspore-mother-cells which is eventually formed. As
the microsporangium enlarges in the spring these cells not only
increase in size but multiply in number. During the last of
March and first of April karyokinetic figures, representing
various stages of division, are seen in all preparations, and in
all cases division is proceeding by the typical method character-
istic of vegetative or sornatic cells. In the latter part of April
or first of May (for Pinus Strobus about the middle of May),
typical division ceases, and, after a period of growth, the pro-
phases characteristic of the heterotypical division are entered
upon. The time at which the rest preparatory to the hetero-
typic mitosis begins varies by about three weeks in the different
species, and by ten or more days in the same species in different
seasons. Had Coulter and Chamberlain examined microspo-
rangia during the latter part of March they would doubtless
have found typic divisions taking place in the archesporial
tissue.
2O MARGARET C. FERGUSON
TETRAD-DIVISION.
The Definitive Arches for -turn. — During the period of " rest "
preceding the heterotypic division, the microspore-mother-cell
increases much in size, its nucleus becoming even larger than
an entire cell of the primitive archesporium, as is readily seen
by comparing figs, i and 2 with figs. 3 and 4. The walls en-
closing the spore-mother-cells thicken considerably, and the
cytoplasm assumes a fine, almost granular structure which,
under high magnification, resolves itself into a delicate, close
reticulum. At this stage, only three or four nucleoli are found
within the nucleus, but this reduction in number may be only
apparent, for the nucleus has enlarged to such an extent that
no one section would be liable to contain as many of these
structures as would a section of one of the smaller nuclei of
the primitive archesporium. No attempt has been made to de-
termine the exact number of nucleoli in the nuclei of the arche-
sporium at any time in its history, as it is next to impossible to
trace accurately the sections in the series of any given cell when
each anther contains hundreds of archesporial cells all of which
are practically alike in form, structure and staining capacity.
As the nucleus of a pollen-mother-ceil enlarges, its reticu-
lum becomes more open, the threads of the net gradually in-
crease in thickness, the net-knots or karyosomes become more
or less prominent, and numerous smaller granules are distrib-
uted irregularly upon the linin. Many cross-threads are with-
drawn but no true spireme is formed at this time (fig. 3). The
thickening of the threads is more prominent in Pinus Strobus
than in the other species, the net-knots are more conspicuous,
and a somewhat imperfect spireme arises, although here, too,
many anastomosing threads still persist (fig. 4). A remarkable
change has taken place in the attitude of the different elements
of the cell towards stains. When the microspore-mother-cells
are first formed both cytoplasm and nuclear net stain more or
less diffusely with gentian-violet as in the primitive arche-
sporium, but, as growth proceeds, the cytoplasm ceases to react
to chromatin dyes and takes the orange G with avidity. The
nucleoli are colored far less deeply with the gentian-violet than
LIFE HISTORY OF PINUS 21
formerly, and the nuclear reticulum takes the blue characteris-
tic of chromatin. In this condition, the contracted state known
as synapsis is entered upon.
The First Nuclear Division of the Microspore-mother-cell.
— As soon as a microspore-mother-cell has attained full size, cer-
tain changes in its nucleus indicate that the prophase of the first
division has been initiated. The reticulum gradually draws
together, its threads becoming thicker and the meshes smaller
(figs. 5 and 6). Contraction continues until the network forms
a compact mass at one side of the nucleus. During synapsis
the nucleoli may be entirely confined within the contracted
sphere or one or more may be partially extruded (fig. 7). Some
of the nucleoli still stain deeply with the gentian-violet, but
one or more usually take the plasma stain at this time and
appear as yellow, porous, or spongy bodies. The same appear-
ance has also been obtained with iron-has matoxylin followed by
orange G.
In Pinus rigida no appearance at all comparable with that
known as synapsis is observed until April 21. In material pre-
served on this date a few nuclei in all anthers show the begin-
nings of contraction as illustrated for P. austriaca in fig. 5 and
P. Strobus in fig. 6. On April 30 the nucleus of every mother-
cell has reached the point of greatest condensation, its contents
forming a somewhat spherical, deeply-staining mass at one side
of the nuclear cavity — fig. 7 illustrates this stage for P. Strobus.
On May 2 some of the nuclei still retain this structure while
others show various stages of recovery. Two days later, May
4, not a vestige of this condition remains, all the nuclei having
passed on to more advanced stages in the mitosis. These
dates have been given for Pinus rigida, but they would not
differ materially in the other species, except that in P. Strobus
corresponding phases in this division would occur about 3 weeks
later.
Synapsis is not universally recognized as a normal step in the
heterotypical division. Guignard ('97), Mottier ('97), Schaffner
('01), and others still look upon it as an artifact caused by im-
perfect fixation. On the other hand, Sargant ('97), Wiegand
('99), Duggar('99 and 'oo), Ernst ('01), Rosenberg ('01) among
22 MARGARET C. FERGUSON
botanists, and many zoologists consider it a definite characteristic
of the early prophase of the heterotypic mitosis, several of these
investigators having noted it in their material before fixation. I
have observed this stage in the fresh material in Pinus, and
after carefully studying it in many permanent preparations, I
see no reason why this condition, simply because it happens to
be one of contraction of the nuclear substance, should be set
down as abnormal.
If this appearance were produced artificially why should
there be transitional forms both in leading up to and in recovery
from it? If it were the result of diffusion currents, as has been
suggested, we should expect to find the nuclear substance in
all the nuclei of a given anther carried or forced to the same
side of the nuclear cavity, but such is not the case. It is doubt-
less true, as indicated by Strasburger ('95), that many phenom-
ena described as synapsis represent pathological conditions
which do not occur under all circumstances, but it seems equally
true that this condition of the nuclear substance represents, in
some species at least, a characteristic stage in the heterotypic
division. Although a contraction comparable with that of
synapsis has been reported for somatic cells, I am not aware
that anything like so marked an appearance has been described
as a usual accompaniment of any but the heterotypical division.
The exact significance of this phase is not well understood,
but that it is intimately associated with a readjustment of the
chromatic and nucleolar substances there can be little doubt.
As the nucleus slowly recovers from synapsis, it soon becomes
apparent that the reticular structure has been replaced by a broad,
closely coiled band which stains more deeply than did the net-
work prior to the contracted stage. The coils of the thread
gradually open out until the nuclear cavity is filled with a
spireme, which consists of a broad linin band, so irregularly
studded with chromatin-granules that it has a much roughened,
almost minutely echinulate, appearance. These granules soon
collect into indefinitely outlined masses which remain connected
by clear, faintly staining portions of the linin thread. The chro-
matin-groups never assume the definite disk-like form figured by
Mottier ('97) for Lilium and Helleborus, and by Duggar ('oo)
LIFE HISTORY OF PINUS
for Symplocarpus, but they remain always irregular and jagged
in outline (figs. 8 and 9). Whether there is one continuous
thread or more than one could not be determined with certainty,
as the coil is at first very densely massed, and free ends might
be obscured. When the loose skein fills the nuclear cavity
more than one spireme can usually be detected, but the indica-
tions are that this effect has been produced by the microtome
knife. At certain places the coils of the spireme run together
and appear to be more or less anastomosed. Such a point of
contact always indicates the position of a nucleolus which has
become almost obscured by the massing of the thread about it,
figs. 9, 13 and 15. Not all the nucleoli are found thus associ-
ated with the skein, but in those cases in which they are free
from the coils of the nuclear thread their capacity for staining
has generally been greatly reduced (figs. 9, n and 15).
As soon as the chromatin-band has become loosely wound
about the entire nuclear cavity, longitudinal splitting occurs,
and the segmentation of the spireme becomes apparent (fig. 10),
but transverse fission is not completed until the longitudinal
division has taken place (fig. n). The segments are long,
coiled, and present various appearances. Whether they
correspond in number to the number of chromosomes eventu-
ally formed, I could not ascertain with any degree of certainty,
since they are so long and closely intermingled in the nucleus
(fig. 1 1). Most of those shown in figs. 1 2 and 1 2 , # , were taken
from sections through the edge of nuclei, and, while they rep-
resent the looped and twisted condition of the chromatic seg-
ments at this time, they have in many instances been cut during
sectioning so that only a portion of most of the segments
appears. From a study of many nuclei containing chromatic
threads similar to these, it is evident that the looped figure has
not been formed by the bending on itself of one of the longi-
tudinal halves of a segment. There are no* indications that the
sister-halves of any portion of the nuclear band ever become
entirely disassociated. They may separate widely at one or
both extremities, but at some point along the thread, an inti-
mate relation is permanently maintained. The loop arises,
therefore, by the complete fusion of the sister-threads at one of
24 MARGARET C. FERGUSON
their free ends (fig. 12, #, c, d, e). Even in such a late stage of
fission as that represented in fig. 13 the sister threads can almost
invariably be traced, but not always, as some are out of focus
and others are doubtless in another section.
The stages immediately following longitudinal splitting and
segmentation of the nuclear spireme are somewhat different
from any that I have seen described by other writers. So
puzzling were they to me when the study of microsporogenesis
was first undertaken in 1899 that a paper, partially prepared at
that time, was laid aside until a larger experience with cell
structures could be brought to bear upon this, which is to me
at once one of the most intricate and interesting problems con-
nected with the activities of the cell. As stated in the intro-
duction, new material was collected in 1901 and fixed with great
care. Many slides were subsequently prepared, and the phases
in the tetrad-division were found to accord perfectly with those
observed during the first period of study. The interpretation
of the phenomena noted is, however, much more satisfactory
now than formerly, although there is still much that is obscure.
Sporogenesis has not been studied in Pinus montana var. un-
cinata^ but there is complete accord, except in such details as
have already been mentioned, in the other four species.
Longitudinal division is scarcely more than completed when
the double skein begins to contract, the two halves of each seg-
ment twisting upon each other to a greater or less degree and
gradually fusing. As the segments contract the sister-halves
may frequently become more or less twisted upon each other ;
they may appear as parallel threads ; the half segments may
separate at both ends, remaining united at the middle only ;
or, having fused at both extremities, they may open out,
forming rings (figs. 12 and 12, a). Fusion invariably begins
first about those nucleoli which have still retained, although
in a less degree than prior to synapsis, the power to react to
chromatin-stains (fig. 13). Contraction and fusion continue
until a coarse, more or less anastomosing structure is formed
in which only traces of the earlier longitudinal division re-
main evident (fig. 14, plate II), and a little later all signs of
fission, both longitudinal and transverse, disappear (fig. 15).
LIFE HISTORY OF PINUS 25
As the thread thickens and broadens it becomes irregular in
outline, the irregularities increase, those from neighboring por-
tions of the threads meeting and fusing. Soon afterwards a
transverse division again becomes apparent (fig. 16). The
segments continuing to shorten and thicken gradually draw
away from one another, finally remaining united only by
delicate threads ; the connecting fibers are at last severed and
the chromosomes lie free in the nuclear cavity. The usual
number of segments formed is twelve, although thirteen, four-
teen, and, in rare instances, as many as sixteen have been
counted (figs. 16, 17, 18, a-c, and 20).
The chromosomes thus arise from an incompletely reticu-
lated structure rather than directly from the spireme. While
this suggests the condition in magnolia where, as recently
described by Andrews ('01), the chromosomes arise directly
from the resting reticulum without the intervention of a spireme,
it is, in matter of fact, very different. We have here not a
nuclear reticulum in the ordinary acceptation of that term, but
a somewhat reticulated structure formed by the anastomosing
with each other, at certain points of contact, of adjacent por-
tions of a previously longitudinally split spireme. As the
chromosomes separate out almost every conceivable form may
be found, not only the X's, Y's and V's of Belajeff, but rings,
parallel rods, eights open and closed, L's, U's and irregular-
shaped bodies (fig. 19, a-l).
In my earlier study of this phenomenon, I supposed the
chromosomes to be the equivalents of the long, coiled segments
first formed, and with such an hypothesis the whole series of
events following longitudinal fission was inexplicable. But after
again considering not only such stages as those represented in
figs. 10-17, but every transitional form connecting them, I am
convinced that this assumption was incorrect and that each seg-
ment consists, rather, of two distinct chromosomes standing
side by side, each half of the double chromosome represent-
ing two sister-segments which were formed by the earlier longi-
tudinal fission but have now fused. If such be the origin of
these chromosomes, and I no longer have any hesitancy in
affirming that they have thus arisen, the phases following the
Proc. Wash. Acad. Sci., July, 1904.
26 MARGARET C. FERGUSON
longitudinal and transverse divisions of the skein are no longer
unintelligible. The sister-threads formed by the longitudinal
splitting not only unite again, but adjacent portions of the
double threads draw together and become more or less fused,
giving rise when transverse fission again becomes apparent
to the one half number of chromosomes. The forms of the
resultant chromosomes are exactly what would be expected
from such an origin. In fig. 18, b, for instance, adjacent
portions of double segments have fused at the ends, trans-
verse division has followed, and three chromosomes — parallel
rods, a U, and a Y, are seen in the act of separation. When
the component chromosomes have fused at both ends only, the
ring, or, if a twist follows, the closed eight results ; if fusion
has occurred at but one extremity the V, U, or open eight is
formed ; if the segments remain attached at the middle point the
X occurs ; when the constituents of the double chromosomes
have united end to end and the bend has not taken place at the
point of their union the L results and so on. The structure or
composition of the X, Y and V forms of chromosomes as found in
plants have been explained in much the same way as the above
by Belajeff ('97 and '98), but he did not trace their development
from the closed spireme and considered these three forms as the
typical or characteristic ones whereas, in Pinus, the other forms
named have been quite as frequently observed. When the
chromosomes first become apparent, irregular fragments of the
chromatic substance are frequently left at various points (fig.
17), but these are ultimately absorbed, doubtless being appro-
priated by the growing chromosomes (fig. 20). l
At the time when the chromosomes are being differentiated,
they often appear as if pulling away from the nucleoli, and may
be seen still connected with them by delicate threads (figs. 18, a
and c). The nucleoli now have a spongy or porous appearance
and fail almost absolutely to take either nucleolar or chromatic
stains. With the final separation of the chromosomes they dis-
appear altogether. The history of these nucleoli from the
primitive archesporium up to the time of their dissolution leads
irresistably to the conclusion that here, at least, there is a very
1 See " Note " at close of Appendix.
LIFE HISTORY OF PINUS 27
intimate relation between nucleolar and chromatic substances.
Whether the nucleoli are actual reservoirs of chromatin which
is given out passively to the chromatic thread, or whether they
are actively engaged in furnishing nourishment to the chromatic
substance, I have not been able to determine, but, from certain
observations to be described in a later chapter, I am inclined to
consider them more than passive elements of the cell.
Coordinate with the formation of the chromosomes the nuclear
membrane resolves itself into a weft of threads which crowd
into the nuclear cavity, together with delicate granular fibers
from the cytoplasm. The latter are evidently formed by a re-
arrangement of the granules of the cytoplasmic reticulum. Up
to this time the cytoplasm has remained close meshed in the
region of the nucleus but has become less dense at the periphery
of the cell. As the nuclear membrane disappears, coarser
reticulations arise in the cytoplasm and extend towards the
nucleus, doubtless contributing to the forming spindle. When
the achromatic figure is fully developed, the cytoplasm again
becomes uniform in structure throughout the cell, but there
seems to have been an actual loss in granular substance, the
meshes of the network being much larger now than formerly
(figs. 20 and 21). A few delicate fibers may be seen in the
cytoplasm just before the dissolution of the nuclear membrane,
but, although I have searched repeatedly for cytoplasmic phe-
nomena such as that described by Mottier ('97 and '98), Duggar
('oo), Juel (Joo) and others, I have never been able to detect
anything at all comparable with the structures figured by these
authors. If they are present in Pmus, I have not been able to
differentiate them with any of the stains used.
The spindle is almost invariably tripolar in origin, but it may
arise as a multipolar diarch. In either case, its ultimate form
is that of a sharply pointed bipolar spindle (Figs. 21-24).
Belajeff ('94) describes this spindle as many poled in origin in
LariX) and Mottier ('97) makes the same statement for Pinus;
but in the many thousands of karyokinetic figures observed for
this division, I have never found one that showed more than
three poles. A few scattering fibers have occasionally been seen
to pass from all sides towards the nucleus but achromatic threads
have not been found to converge at more than three points.
28 MARGARET C. FERGUSON
As the spindle-fibers press into the nuclear cavity, the chro-
mosomes take up their position at the equatorial plate. They
are now very regular in outline, apparently homogeneous, and
the X, Y, V, O, etc., forms can still be clearly distinguished
(fig. 24, plate III). Each segment is oriented with its longer
axis perpendicular to the axis of the spindle, the free limbs ex-
tending outward. The spindle-fibers are attached at one ex-
tremity of the parallel rods, and ordinarily at or near the point
of union of the constituents of the dual chromosomes. In the
Y-shaped chromosomes the achromatic threads may become at-
tached at the point where the two limbs become free or at the
free end of the fused chromosomes, but, whatever the shape of
a segment, the spindle-fibers are never attached at the extremi-
ties of its free limbs.
The line of cleavage at the equatorial plate is not such as
to separate the two chromosomes but is rather such as to effect
a longitudinal splitting, the two half chromosomes of each pair
passing together to opposite poles. During metakinesis the
daughter-chromosomes become very irregular in outline and in-
crease much in size, the half chromosomes apparently exceeding
in volume the undivided ones (figs. 25-28). This augmentation
of the segments maybe due to actual addition of new substance,
but from the fact that in the telophase they are unquestionably
smaller than in the late prophase, it is probable that this is
merely an amplification without actual or permanent growth.
The parts of the spireme separated during the longitudinal fis-
sion following synapsis have so completely fused again that
they are now disunited with difficulty. The appearance of the
dividing chromosomes indicates that they are being subjected
to great strain. Under this tension they are flattened out and
rendered irregular in outline ; the irregularities result from the
unequal stretching of the chromatic substance at different
points, just as a poor rubber band when greatly extended be-
comes more or less moniliform. The complete separation of
the half chromosomes may sometimes be greatly delayed, when
the stretched segments extend nearly the entire length of the
spindle, the achromatic figure being almost obscured, in some
instances, by the chromosomes (figs. 25, 26, 28 and 29). That
LIFE HISTORY OF PINUS 29
these segments are actually flattened out is further shown by
the fact that the arms which remain united and elongated stain
much less deeply than do those which, having become free,
have contracted to nearly their former length. This would
seem to indicate that the chromatic spireme is a plastic or viscid
body. Lloyd ('02) describes a similar action, though much
less marked, in Crucianella. While the position of the retreat-
ing half chromosomes is such as to give ordinarily the appear-
ance of V's or U's, other figures occur with sufficient frequency
to establish the reality of their persistence after the close of the
metaphase of the division. This point will be considered more
fully later.
The achromatic figure increases but little in length as the
chromosomes pass to the poles so that the movement here must
be due in large measure to a pull exerted by the contracting
fibers and not to any great extent to a push brought about by
the growth of the central spindle. If the force which seems
necessary to effect the separation of the half chromosomes is
furnished by the achromatic fibers, we should expect to find the
poles of the spindle firmly buttressed as described by Stras-
burger ('oo) for Larix; but no strengthening fibers are devel-
oped, and, although the apices of the spindle are usually
inserted in the ectoplasm, they not infrequently end blindly in
the cytoplasm. It is possible that the force exercised by the
growing fibers of the central spindle just equalizes the counter
force exerted by the mantle fibers in drawing the chromosomes
to the poles, the equilibrium thus established giving rigidity and
rendering a support for the poles unnecessary. By the time
the pairs of daughter-chromosomes have reached the poles they
have become much reduced in size and regular in contour (figs.
27 and 30).
After the chromosomes reach the point where the daughter-
nuclei are to arise, they do not at once fuse end to end to form
a continuous spireme, but as the chromosomes lie side by side
they lose their clear outline and gradually assume a diffuse
reaction to stains. In this condition the halves of the longi-
tudinally split pairs of chromosomes are doubtless fused, after
which fusion the adjacent segments unite by their ends to
30 MARGARET C. FERGUSON
form a coiled, somewhat moniliform thread (figs. 30-32).
Immediately upon the formation of the skein a delicate
nuclear membrane appears, the coils loosen somewhat and
branch freely thus giving rise to a reticulum. Extensive
growth follows and a large " resting" nucleus is formed (figs.
33 and 34). The nuclear net consists at first of delicate achro-
matic linin threads bearing scattered chromatin-granules and
uniting large irregularly branched chromatic portions. Distri-
bution of the chromatin continues until there is a delicate linin
reticulum with chromatin granules of varying sizes imbedded
in it (figs. 33-35). These nuclei have the form of a plano-
convex lens the flat side of each nucleus being perpendicular
to the axis of the spindle and facing the other daughter-nucleus.
It is obvious from the foregoing that a definite resting nucleus
is formed in Pinus at the close of the heterotypic division.
This accords with the recent observations on the formation of
the microspore by Duggar ('99) in Bignonia, Strasburger (5oi)
and Gager ('02) in Asclepias and Andrews ('01) in Magnolia.
A true nucleolus has not been observed in the daughter-nuclei.
Contrary to the observations of Hofmeister ('51), no cell-wall
is laid down and in only a very few instances has a slight
thickening of the spindle fibers in the region of the cell-plate
been observed.
The Second Mitosis of the Mother-cell. — The resting daugh-
ter-nuclei are scarcely more than established before the initial
steps of the second division are instituted, as evidenced in the
readjustment of the nuclear reticulum. The more delicate
threads of the net are withdrawn, the nuclear membrane fades
out, the chromatin loses its granular aspect and becomes evenly
distributed upon the linin, and there issues forth a heavy, homo-
geneous, deeply-staining band which is more or less coiled and
branched (fig. 36). The chromatin-thread, which now lies
free in the cytoplasm of the mother-cell, continues to thicken,
the branches or cross fibers disappear, and in an almost incredi-
bly short time, the delicate nuclear net has given place to a
broad, somewhat spirally coiled skein (fig. 37).
Achromatic threads arise in the cytoplasm forming a multi-
polar diarch spindle. The fibers are not abundant and always
LIFE HISTORY OF PINUS 3!
arise in a plane perpendicular to the axis of the primary spindle.
Harper ('oo) makes the statement that in Larix, where no cell-
wall follows the first division of the pollen-mother-nucleus, the
spindle-fibers of the primary mitosis are utilized in the formation
of the spindle for the second division. I am unable to trace any
such connection in the pollen-mother-cells of Pinus, all traces of
the first karyokinetic figure having been lost to view before the
inception of the spindle for the second division.
As the kinoplasmic fibers appear the chromatin-band forms a
double row of loops extending across the spindle-threads in the
plane of the equatorial plate. The longitudinal splitting is now
clearly apparent. The loops continue to shorten, and in this
position transverse fission occurs, segmentation almost always
taking place at the outer free ends of the loops (figs. 38 and 39,
plate IV). The sister-halves of each V- or U-shaped chromo-
some entirely separate, undergo readjustment, and finally come
to stand in a double row with their free ends in the line of the nu-
clear plate and their angles towards their respective poles (figs.
38-41). The spindle-fibers become attached to the chromosomes
at their point of bending, and the half chromosomes pass to the
poles (figs. 42-43). The dissociation of the sister-halves of each
segment is so complete before the beginning of the separation
at the equatorial plate that the figure during metakinesis is such
as to give the impression of whole chromosomes passing to the
poles, but a study of the prophases of the division shows clearly
that each represents the half of a double chromosome. In the
telophase of the division the chromosomes unite end to end to
form a spireme(fig. 44). The nuclear membrane appears, and
the chromatic band branches, giving rise to the reticulum of
the resting nucleus (figs. 44 and 45).
The Problem of Reduction. — Here as in all studies of spore-
formation at the present time the question of reduction demands
consideration. As already indicated, the reduction in the num-
ber of chromosomes takes place, as is the rule, during the so-
called resting stage of the spore-mother-cell, the one half num-
ber of chromosomes appearing in the prophase of the hetero-
typical division. But the inquiry concerning the presence or
absence of a qualitative reduction is not so easily answered.
32 MARGARET C. FERGUSON
With few exceptions, botanists of to-day follow the present
lead of Strasburger and accept the view of a double longitudinal
splitting of the chromosomes in the first division of the spore-
mother-cell. According to this interpretation, reduction, in the
sense in which Weismann uses the term, does not occur in
plants. Among the exponents of a qualitative or true reduction
in plants, Atkinson ('99), Belajeff ('97, '98), Calkins ('97),
Ishikawa ('97, '01), and Schaffner ('97, '01) are almost alone
to-day in not having retracted their earlier published conclusions
regarding this subject.
It has seemed best to record the details of the observations
made in studying the tetrad-division in Pinus, before entering
upon any discussion of the significance of the phenomena noted,
but in so doing some reiteration is inevitable.
Strasburger's statement that certain forms of chromosomes
occurring in the anaphase of the heterotypic division are inex-
plicable on any other assumption than that of a double longi-
tudinal splitting is, doubtless, correct when those forms have
been derived from V-shaped chromosomes. But, while it may
be true that such figures are due to a double longitudinal fission
when derived from other than V-shaped chromosomes, it is like-
wise true that, in such cases, the phenomena are capable of
rational explanation on other grounds. The V with the three
arms, for instance, may result from the attachment of the spindle
fibers at the middle point of a Y, the stem of the Y bending
down as it moves to the poles (fig. 30, <z, plate III), and a double
V might be derived in the same way from an X-shaped chromo-
some (fig. 30, c). In fig. 26 the second chromosome on the left
represents a Y opening out from its lower extremity, and the next
chromosome shows parallel rods just separating. Occasionally
an X or Y figure becomes apparent in the late anaphase of this
division (figs. 28, 29). Such appearances are doubtless to be
attributed to an early straightening out of the segments. If the
constituents of the double chromosomes are disunited in this
mitosis, then such chromosomes as those illustrated in figs. 28,
d, and 30, <z, c, and e, might result from the more or less com-
plete longitudinal fission of the sister-segments. Should this
prove to be the case, and if my interpretation of the origin of
LIFE HISTORY OF PINUS 33
these chromosomes is correct, then both a quantitative and a
qualitative reduction of the chromosomes would occur in the
first or heterotypic division, and whole chromosomes, each
representing the half of a dual chromosome, would pass to
opposite poles. I am aware that such a phenomenon has been
described by Atkinson and a few others, but after long and care-
ful study there does not seem to me the least doubt, that, in the
case of the pines investigated, a longitudinal fission, and not a
transverse one, occurs in this first mitosis ; and X-, Y-, and
ring-shaped segments, as well as V's, pass to the poles, although,
as Belajeff has pointed out, they usually, because of their posi-
tion, have the form of V's in the anaphase of this divison.
Most writers on sporogenesis, and especially those who are
advocates of the true reduction, have not found a resting nucleus
intervening between the heterotypic and the homotypic divisions.
As already stated a resting nucleus is clearly demonstrated at
this point in Pinus. The spireme formed from this nucleus
shows signs of longitudinal division before segmentation, and,
while lying at the equatorial plate, the two halves of each seg-
ment separate entirely, in most instances at least, before their
final orientation on the spindle. Now the question arises as to
whether or no this homotypic division effects a qualitative reduc-
tion . If the theory of the so-called * ' individuality of the chromo-
somes " is without foundation then it certainly does not ; but, if
the possibility of the complete rehabilitation of the chromosomes
be accepted, a qualitative reduction very probably does occur.
For under such conditions, the skein preceding the homotypic
division would consist of the daughter-chromosomes, formed as
a result of the heterotypic mitosis, fused end to end. These
daughter-chromosomes, it will be remembered, arose by the
longitudinal fission of a double chromosome and each, therefore,
consists of a pair of half chromosomes. Thus the second,
apparently longitudinal, splitting would effect the separation of
the half chromosomes of each pair, rather than the longitudinal
fission of a single chromosome. Reduction would thus take
place in the true or Weismann's sense. Because of certain
phenomena to be described in connection with the development
of the pro-embryo, I am inclined to believe that the chromo-
34 MARGARET C. FERGUSON
somes retain their individuality through succeeding cell-genera-
tions. I am, therefore, disposed to regard the tetrad-division
in Pinus as a true reducing division ; in this way only does the
complicated process just described find satisfactory explanation.
No positive statement can, however, be made either way, in
connection with this division in Pinus, until we are in posses-
sion of greater knowledge than at present of the origin and ulti-
mate destiny of chromosomes.
Guignard ('97) expresses the opinion that the regularity of the
chromosomes in certain forms has been overestimated. Be that
as it may, I am conscious that there is recorded in this paper a
greater variation in the forms of the chromosomes than has been
described in a single genus by other writers. It has been my
purpose to note not only that which is in accordance, or at
variance, with the observations of other investigators, but to
give as faithful a record as possible of the conditions found in
the preparations studied. And may we not yet find that here,
in the divisions preceding spore-formation in plants, as in many
other instances, there is greater variation in matters of detail
than was formerly supposed to be the case?
DEVELOPMENT OF THE MICROSPORE.
The Formation of the Spore-wall. — Hofmeister ('51) de-
scribed four " special " cells, each with its own wall, within the
pollen-mother-cell in the AbietinecR^ and Juranyi ('72 and '82)
devoted particular attention to the formation of the wall of the
microspores in many Gymnosperms and Angiosperms. He
described the development of a wall separating the two nuclei
after the first division. This wall was soon absorbed and during
the second division the entire cell was filled with connecting
fibers stretching between the four nuclei. Delicate walls were
then laid down between the nuclei giving rise to the four
microspores. These dividing walls thickened and united with
the inner wall of the spore-mother-cell ; thus a portion of each
spore-wall was formed from the inner mother-wall. After a
period of rest the outer mother-wall was burst and the " pollen-
cells " became free. If there is any recent literature of value
on this subject, I have failed to find references to it.
OF T nt *
UNIVERSITY
LIFE HISTORY OF PINUS 35
As already indicated, no wall separating the daughter-nuclei
is formed at the close of the heterotypical division in Pinus.
During the late telophase of the second mitosis in the microspore
mother-cell, a readjustment of the spindle-fibers occurs giving
rise to the complex figure that has been described as character-
istic of spore-formation in many plants. The development of
the archoplasmic structures connecting the nuclei of the tetrad
is much less marked than in Podaphyllwn (Mottier '97) and in
many other phanerogams (fig. 44). By the time the nuclei
have reached the resting stage, a division has occurred in the
cytoplasm giving rise to four cells which are surrounded by
delicate clear walls. A prominent thickening of the wall of the
spore-mother-cell takes place, and at the same time a thick wall,
continuous with the inner portion of the mother-wall, appears
between the daughter-cells.
This wall frequently attains remarkable thickness. Whether
it constitutes an inner wall, or is merely a thickening of the
primary wall by the deposition of new material on its inner sur-
face, I am unable to say. The outer, primary wall stains more
deeply and is frequently seen separated from the inner broad
portion (figs. 44-47). This inner wall, which is continuous with
the broad walls separating the young microspores, stains deep
yellow with orange G, if the orange is allowed to act from
one to two minutes ; it appears a pale rose when treated with
safranin, but fails altogether to stain with iron-hasmatoxylin.
In a few instances, slight evidences of stratification have been
observed, but ordinarily the wall appears perfectly homogene-
ous, giving the impression of a liquid or viscid substance in
which the spores are imbedded ; but the fact that it is often
separated from the outer wall by a clear space, and also that it
is left behind as a definitely outlined wall after the escape of
the spores militates against the probability of its fluid nature.
After the spores have grown for a certain period the mother-
wall is ruptured and the spores are liberated. At this time the
empty mother-cell with its four chambers is often met with
(figs. 48, 49).
In so far as I am aware, this permanent division of the
mother-cell into four compartments by thick cellulose walls has
36 MARGARET C. FERGUSON
not been previously described. A broad open space, repeatedly
figured between the daughter-spores and the mother-wall, has
been invariably attributed to shrinkage ; but it is probable that,
in some cases at least, it represents this thickened wall which
has failed to be differentiated with the stains used. Wiegand ('99)
says that the spores of Potamogeton are as if imbedded in a
ground mass of some viscid substance, but he does not figure it
and makes no statement regarding the development of cell-walls
between the microspores.
Origin of the Air-sacs. — As soon as the young microspores
have become enclosed, each within its own special chamber of
the mother-cell, it is evident that a special wall has been de-
veloped about each spore. This is doubtless secreted by its
own cytoplasm and is not, as Juranyi thought, derived from the
inner wall of the microspore-m other-cell. The spore-wall while
still very delicate becomes differentiated into an inner and an
outer layer corresponding to the intine and extine of the pollen-
grain. The young microspores are characterized by the rela-
tively large size of their nuclei, the nucleus filling almost the
entire cell just prior to the discharge of the spores. The cyto-
plasm which fills the remainder of the cell is in the form of a
loose reticulum (figs. 46, 47).
As time goes on the outer wall of the microspore expands at
two points on opposite sides of the spore. A resistance is met
with in the thick wall of the spore-mother-cell and the plastic
inner wall of the microspore responding to this new pressure
becomes indented along the surfaces corresponding to the ex-
tended portions of the outer spore-wall. Thus a clear open space
having in section the form of a biconvex lens is formed between
the extine and the intine on either side of the microspore.
These are the beginnings of the wings or air-sacs that are so
conspicuous in the mature pollen-grain of the Abietinece.
Finally the pressure becomes so great that the mother-wall is
ruptured and the spores are liberated (figs. 47, 48). Coulter
and Chamberlain ('01) noted the fact that the wings make their
appearance in Pinus Laricio while the microspores are still
within the mother-cell, but they recorded no observations regard-
ing the origin and development of these sacs. Strasburger and
LIFE HISTORY OF PINUS 37
Hillhouse ('oo) consider that these bladder-like appendages con-
sist of the outer part only of the extine, the extine having under-
gone cleavage at these two points. In studying the develop-
ment of these organs from their earliest beginnings, it appears to
me that the line of cleavage lies rather between the two coats of
the young spore. If it is not, then at the time that the micro-
spore leaves the parent-cell, the intine has not been developed,
or, if present, is so delicate that I have not been able to detect
it (fig. 48).
Growth of the Micros-pore. — After its escape from the
mother-cell the microspere undergoes rapid growth, and the
outer surface of the spore becomes beautifully marked by the
formation of delicate, irregular ridges over the entire inner sur-
face of the extine, except along that portion which connects
the two wings on the concave or ventral side of the pollen-grain.
It is at this point that the pollen-tube later makes its exit, and
there is here no appreciable thickening of the spore- wall. These
ridges continue to grow and extend inward forming a very pretty
reticulated structure which is most distinctly apparent on the
walls of the wings ; along the convex or dorsal side of the
pollen-grain the reticulations are closer and the extine forms a
broad, deeply staining layer (figs. 50-54, plate V). This
irregular thickening of the extine is an admirable adaptation for
securing strength with slight increase in weight.
When the young microspore attains to its mature-size, a par-
tial wall, extending along the back and for a longer or shorter
distance down the sides of the spore, becomes apparent within
the intine (fig. 54). It consists of a broad, homogeneous-
appearing band which gives precisely the same staining reac-
tions as the thick wall developed within the spore-mother-cell
after the formation of the young microspores. These immature
pollen-grains, after treatment with Flemming's triple combination
or with the gentian-violet and orange G alone, afford the most
brilliant effect that I have observed with these stains. The extine
presents a very intense, clear blue, the inner homogeneous wall
an equally vivid yellow, while the protoplasmic elements take
the colors characteristic for these dyes. The fact that this third
partial wall fails entirely to respond to some stains doubtless
38 MARGARET C. FERGUSON
accounts for its having been overlooked by previous writers. It
is not shown at all in the series of figures, recently published by
Coulter and Chamberlain ('01), illustrating the development of
the pollen-grain in Pinus Laricio.
The various tests commonly used in determining the nature
of the cell-wall have been applied to the young pollen-grains
as well as to the special spore-mother-walls. These tests show
that the outer wall of the pollen-grain is clearly of the nature
of cutin, as has been demonstrated by Strasburger. Both the
innermost wall of the microspore, and of the pollen-grain, as
also the wall of the special spore-mother-cells, respond to the
reaction for cellulose, but not in a very marked manner. If
they are of the nature of cellulose there would seem to be an
admixture of some other substance, but I have not succeeded
in obtaining entirely satisfactory results regarding the nature of
these inner, prominent walls. Tests thus far have been made
with " fixed" material only; further experimentation along
this line will be made when fresh material is at hand.
During the season of growth, the nucleus of the microspore
always remains close against the convex or dorsal side of the
spore, occupying a central position along this wall. As is
usual in cell-development, the microspore-cell attains full size
before any mitoses occur within it, and there is never any fur-
ther increase in the size of this cell after the inception of the
first division. The fully developed microspore is, therefore,
the exact counterpart, so far as size is concerned, of the mature
pollen-grain. Compare fig. 54, plate V, with fig. 65, plate
VI. During the development of the microspore, the cytoplasm
which at first was uniformly distributed in a rather loose net
work, becomes more closely reticulated and at the same time
less abundant in proportion to the size of the cell. At the
maturity of the spore the cytoplasm is largely distributed about
the nucleus from which strands extend outward in a radial man-
ner and end in the ectoplasm. In 1898 the microspores of
Pinus Strobus were ready to leave the mother-cells on May 30,
they had attained full size on June 7, and on June 10 the pollen-
grains were fully mature.
LIFE HISTORY OF PINUS 39
SUMMARY.
In Pinus rigida, P. austriaca and P. resinosa the primitive
archesporium is well developed before the approach of winter,
but the microspore-mother-cells do not arise until the end of the
following April. The male inflorescence does not appear in
Pimis Strobus, until the end of the April preceding pollination,
and the definitive archesporium is differentiated in this species
about the middle of May. The nuclei of the primitive arche-
sporium are characterized by several deeply staining nucleoli
and a fine, close-meshed reticulum which responds but slightly
to chromatic dyes.
The wall of the pollen-sac consists in all cases of from three
to four layers of cells. The tapetum is not clearly distinguished
until spring and there are indications that it may be derived
from the outer layer of sporogenous tissue. The nuclei of
this tissue multiply mitotically and the cells reach their maxi-
mum size about the time when the microspores become free.
At this period each cell has from one to three nuclei which pre-
sent all stages of fusion. When the pollen-grains are mature
the tapetum has entirely disappeared and the wall of the micro-
sporangium consists of a single layer of cells, or at most of
not more than two.
Synapsis is recognized as a normal stage in the prophase of
the heterotypical division in the pollen-mother-cell of Pinus.
It is not preceded by a definite spireme, but a broad skein con-
taining irregular masses of chromatin separated by clear portions
of the linin thread issues from the contracted nuclear mass.
The chromatic spireme splits longitudinally and breaks up
by transverse fission into several segments. The loosely coiled,
delicate threads resulting from the longitudinal division soon
draw together and fuse, double threads also come into contact
at various points and fuse more or less perfectly. These threads
always anastomose most freely in the region of the nucleoli,
some of which still stain deeply while others stain but faintly
after synapsis.
Fission occurs at various points in the now irregularly con-
tracted and anastomosed threads, and the separate chromosomes,
40 MARGARET C. FERGUSON
in the reduced number, become apparent. These segments are
at first irregular and jagged in outline showing distinctly the points
at which each has separated from neighboring segments, but they
gradually diminish in size and become more regular in contour.
The chromosomes thus formed are in the form of X's, Y's, V's,
U's, L's, parallel rods, rings, and indefinitely-shaped bodies.
Each segment consists of two chromosomes fused side by side.
The spindle-fibers arise both from the nuclear membrane
and from the cyto-reticulum. The achromatic figure may
originate as a multipolar polyarch of three poles or as a broad
multipolar diarch spindle. At the close of the prophase of the
heterotypic division the spindle has become sharply bi-polar
and its extremities may be imbedded in the ectoplasm or they
may end blindly in the cytoplasm.
The chromosomes are separated at the equatorial plate with
difficulty giving the appearance of a plastic substance under
tension. Their separation may be so delayed that the daughter-
chromosomes stretch from pole to pole. They ordinarily have
the form of V's or U's during the anaphase of the mitosis, but
other forms are not infrequent. The first division effects a
longitudinal splitting of the chromosomes into daughter-seg-
ments of the same form as the parents.
A resting nucleus is established at the close of the first mitosis
but the daughter-nuclei are not separated by a cell-wall. The
daughter-reticulum soon gives rise to a more or less spirally
coiled chromatic band which loops itself at the equatorial plate
and splits longitudinally before segmentation.
The chromosomes have the form of U's and are oriented at
the equatorial plate in two rows with their free ends touching
and the bent portion of each segment directed towards the poles,
the complete fission of the segments having been completed
before their migration to the poles begins. The writer inclines
to the view that these are the half chromosomes of the daughter-
pairs which were separated in the first division. If this hy-
pothesis be correct, the homotypic mitosis in Pinus effects a
true or qualitative reduction of the chromosomes.
The wall of the microspore-mother-cell increases markedly
in thickness and its protoplasmic contents is separated into four
LIFE HISTORY OF PINUS 4!
parts by prominent cross walls which are continuous with the
inner portion of the mother-wall. The microspores are then
developed each in its own particular chamber of the mother-
cell.
A double wall is quickly developed about each spore and the
air-sacs become apparent while the spores are still within the
mother-wall. They arise by the separation of the extine from
the intine at two definite points on opposite sides of the spore.
By the growth of the spore, and more especially by the expan-
sion of the air-sacs, the spore-mother-wall is ruptured and the
spores set free.
Growth ensues, the extine becomes irregularly thickened on
its inner surface except at the concave side of the spore, and a
broad partial wall is laid down just within the intine and along
the back and sides of the microspore. During the growth of
this cell its nucleus maintains a position at the central point of
its dorsal side. Before the germination of the microspore it
attains to the full size of the mature pollen-grain.
CHAPTER II.
THE MALE GAMETOPHYTE.
THE DEVELOPMENT OF THE POLLEN-GRAIN.
Formation of the Prothallial Cells. — So much confusion
has arisen in the application of terms used to designate the
various cells of the male gametophyte in Gymnosperms that it
is desirable, if not almost necessary, that one should define at the
outset the nomenclature adopted. Throughout this paper, the
first two cells cut off from the larger cell are known respectively
as the first and second prothallial cells, and the third small cell
formed represents the antheridial or third prothallial cell. The
large cell, so long as it continues to divide, is designated as the
apical cell, but after division ceases in this cell it is referred to
as the tube-cell and its nucleus constitutes the tube-nucleus.
The antheridial cell divides to form the stalk-cell and the gen-
erative cell, the latter giving rise to the binucleated sperm-cell.
Proc. Wash. Acad. Sci., July, 1904.
42 MARGARET C. FERGUSON
As soon as the microspore has reached maturity, there arises
within its nucleus one of the most beautiful, homogeneous,
loosely-looped and coiled spireme-bands that I have ever seen
in any dividing nucleus (fig. 54). The material studied showed
every stage in the first division, and all succeeding mitoses
which occur within the microspore, but they offer nothing
especially instructive from a cytological point of view, since
they conform to the typic method of division. I shall, there-
fore, describe and figure only such phases as are of interest in
tracing the development of the pollen-grain. It is interesting
to note that in the late prophase of all the mitoses which occur
in the development of the male gametophyte the achromatic
figure presents a very characteristic appearance, being sharply
monopolar at its outer or lower extremity and broadly multi-
polar at the opposite end. It thus forms a fan-shaped body
rather than one resembling a spindle. During the telophase it
usually becomes bluntly bipolar, though the upper pole often
remains to the last somewhat broader than the lower pole (figs.
55, 56 and 60, and plate V. A similar method of karyokinesis
has been noted by Wiegand ('99) in the development of the
pollen-grain in Potamogeton, by Duggar ('oo) in Symplocarpus,
and by Coker ('02) in Podocarpus. This mode of division will
be referred to again in connection with certain phases in the
development of the female gametophyte.
In all the divisions which occur within the wall of the micro-
spore the nuclear substance is divided equally, the cytoplasm
unequally. The nucleus of the first prothallial cell, however,
never equals in size that of the apical cell and always stains
more or less diffusely, thus showing signs of disintegration from
the time of its organization (fig. 57). Fig. 58 shows one of the
very largest and most nearly normal of all the prothallial cells
observed. The nucleus of the apical cell enters the complete
resting stage, instituting a definite network within the meshes of
which one or more faintly staining nucleoli become apparent,
but this reticulum at once resolves itself into a homogeneous,
spireme exactly similar to the one first formed. When the
nucleus of the apical cell has reached the spireme-stage of the
second division, the first prothallial cell is invariably found
LIFE HISTORY OF PINUS 43
pushed against the dorsal side of the spore-wall, not a vestige
of its cytoplasm is left, and the nucleus has become greatly
flattened, although there is still a faint suggestion of its former
reticular character (fig. 59). When the telophase of the divi-
sion is reached this nucleus has lost all traces of its former
structure and persists only as a deeply staining, linear body
lying against the spore-wall (fig. 60). During the following
division it becomes scarcely more than a line so that it is fre-
quently detected with difficulty. Coulter and Chamberlain
('01) figure this cell in Pinus Laricio as still projecting into the
cytoplasm of the apical cell when that cell is in the telophase of
the second division, but I have never found it in such a state of
preservation at so late a date. The second prothallial cell is
invariably smaller than the first, and during the third mitosis of
the apical cell, which follows immediately the formation of the
second prothallial cell, it exactly repeats the history of the first
cell (figs. 61-63).
The partial, broad, innermost wall, described in connection
with the development of the microspore, persists throughout the
entire history of the pollen-grain, and a comparatively broad
wall, continuous with it and having exactly the same staining
capacity, invests both the first and second prothallial cells as
shown in figs. 57-63. The presence of the remnants of the
prothallial cells imbedded apparently in the inner wall of the
mature pollen-grain (fig. 63) was very perplexing before the
history of these cells was studied. But in tracing their develop-
ment it is clearly demonstrated that the remnant of each cell is
pushed back against the wall of the spore and remains perma-
nently covered on its outer side by its own wall. That the
remains of these cells come to lie nearer the intine than when
first formed would again suggest the somewhat plastic nature
of the partial or incomplete membrane against which the pro-
thallial cells are pressed (figs. 57-64). These observations con-
firm the statement of Strasburger, Noll, Schenck and Schimper
('97) that the two prothallial cells formed in the pollen-grain of
the Gymnosperms are invested with cellulose-walls. Coulter
and Chamberlain ('01) make no mention of the formation of
walls in connection with the development of these cells in Pinus
44 MARGARET C. FERGUSON
LariciO) and Coker ('02) says that in Podocarpus " as in other
cases" no cellulose-wall is formed. The small cell cut off by
the third and last division of the apical cell persists as a perma-
nent feature of the mature pollen-grain. Its cytoplasm is dis-
tinctly differentiated from that of the tube-cell, but no cellulose-
wall has been observed in connection with this cell, its boundary
being marked by scarcely more than a condensation of its periph-
eral cytoplasm.
The Mature Pollen-grain. — During the development of the
male gametophyte the cytoplasm of the large cell gradually
increases in amount, the vacuoles becoming smaller from the
region of the nucleus outward, and finally disappearing alto-
gether. The pollen-grain has the same size, form, and, so far
as the wall is concerned, the same structure as the microspore
just prior to its germination. The thick, innermost, partial wall
described in connection with the microspore still persists as a
very prominent characteristic of the mature pollen-grain. With
the expansion of the wings, certain protoplasmic portions of the
microspore-cell are left with no support except the delicate endo-
spore ; it therefore seems probable that this broad, incomplete
wall extending along the back and down the sides of the pollen-
grain has been developed for the purpose of strengthening these
weakened points in the spore-wall, and as an additional support
to the dorsal side of the pollen-grain.
But, while the wall of the mature pollen-grain is identical
with that of the microspore, the essential or protoplasmic part
of the spore has undergone marked changes, as we have
already seen. One or two deeply staining lines, more often
one than two in the mature pollen-grain, lie on the dorsal side
of the pollen-grain apparently imbedded in its innermost wall.
Extending from this wall at its middle point is a strongly convex
cell, the antheridial cell, with delicately reticulated cytoplasm
and a comparatively large nucleus. Just below and always in
contact with this cell is the nucleus of the tube-cell. The cyto-
plasm of the tube-cell is closely reticulated and slightly more
dense than that of the antheridial cell. Imbedded in its cyto-
plasm are numerous starch-grains. In this condition the pol-
len-grain of Pinus awaits pollination (figs. 64, 65, plate VI).
LIFE HISTORY OF PINUS 45
Starch-grains have been found in the large cell from an early
date in the development of the pollen-grain, but they are more
abundant after maturity is reached than at any previous time.
According to Coker ('02) the pollen-grains of Podocarpus con-
tain large starch-grains from the beginning of the first division.
With such variations in details as have been noted above, this
description of the development of the pollen-grain in Pinus
agrees with that given by Strasburger in 1892 and Coulter and
Chamberlain in 1901.
POLLINATION.
The Ovule at the Time of Pollination. — In the vicinity of
Cornell University, 42^° north latitude, the pollen-grains of
Pirius Strobus are ready for dispersion late in May or early in
June, but in the other species studied pollination takes place
during the latter part of May. At this time the axis of the
female cone elongates, thus separating the ovuliferous scales
which now make an angle of about thirty-five degrees with the
rachis. After pollination the fruit scales draw together and,
according to Strasburger and Hillhouse ('oo), their edges are
consolidated by the ingrowth of papillae. The presence of two
ovules at the base of each scale, each ovule with its apex extend-
ing downwards, that is towards the base of the scale, and out-
wards, is too familiar a fact to need more than a passing men-
tion here.
As pointed out by Hofmeister ('62) the integument is con-
tinued above the nucellus into two long arms which curve out-
ward before pollination and lead below to a wide mycropylar
canal. The degree of development which the ovule has obtained
at the time when the pollen-grains reach the nucellus is shown
in fig. 66. Deep within the central portion of the ovule, at its
chalazal end, a single cell is distinguished from the others by its
greater size and larger nucleus, this is the macrospore * of Hof-
meister ('51). The so-called "spongy "tissue of Strasburger
is already well differentiated when pollination takes place (figs.
66, plate VI, and 124, plate XII). Somewhat later the integu-
JIn 1901, I stated that, at the time of pollination, there was in the nucellus
an axial row of cells. I know, now, that this condition has rarely been reached
at so early a date, and should be noted as very exceptional rather than as normal.
46 MARGARET C. FERGUSON
ment has closed over the pollen-grains and the macrospore
mother-cell has divided giving rise to an axial row of cells the
lowest of which becomes the functional macrospore (fig. 69,
plate VI).
The Pollen- chamber. — The pollen-grains fall upon a scale
and slip down to its base where they come into contact with the
extended arms of the ovule. These prolongations of the integu-
ment now straighten and partially draw together thus bringing
the pollen-grains down into the wide micropylar canal (fig.
123, plate XII, and fig. 66, plate VI). The free limb of the
integument is seen in section to consist, at this time, of three
layers of cells. As soon as the pollen-grains have found their
way into the lower portion of the micropylar canal and some,
at least, have come into contact with the tip of the nucellus,
the cells constituting the middle layer of the arms, at a point
slightly above the apex of the nucellus, elongate rapidly. The
bulge or protuberance thus formed extends inwards from all
sides and meets, closing the opening above the pollen-grains
(figs. 66 and 67). As soon as the opening has been closed
and the pollen-grains secured, these elongated cells give
rise by division to many smaller ones (fig. 68). By the rapid
elongation of these cells the safety of the pollen-grains is as-
sured in a very short time, and then cell multiplication follows
leisurely. This very pretty mechanism by which the final clos-
ing of the micropyle is effected has not been previously described
for any Gymnosperm, unless it be noted in Shaw's ('96) state-
ment, unaccompanied by figures, that the micropyle in Sequoia
is closed by the radial elongation of the cells about it.
The depression in the apex of the nucellus in the Abielinea
at the time of pollination, described by Hofmeister in 1851, and
since noted by many writers, has, it seems to me, been greatly
exaggerated so far as Pinus is concerned. The expression
" cup-like depression " is not infrequent in literature, but, in so
far as my observations go, saucer-like is as strong a term as one
is justified in using (figs. 66, 67 and 69, plate VI, and 75, plate
VII). At the time of pollination the upper concave portion of
the nucellus terminates in a row of more or less elongated
cells, which are not closely united at their free extremities, but
LIFE HISTORY OF PINUS 47
stand up, as it were, like so many fingers to catch the pollen-
grains ; they also serve to facilitate the entrance of the pollen-
tubes into the tissue of the nucellus (fig. 75, plate VII). A little
later this depression may become more prominent, both by the
slight disintegration of some of the superficial cells of the nu-
cellus, due to the action of the pollen-tubes, and by the incon-
siderable growth, after pollination, of the peripheral layer of
cells of the nucellar tip. The deep cup-like depression some-
times observed is invariably the result of abnormal disintegration.
The pollen-chamber in Pinus, then, consists of a space bounded
on the bottom by the more or less concave upper surface of the
nucellar tip, and arched above by the ingrowth of the free por-
tion of the integument. Later a resinous substance is secreted
which securely seals the opening by which the pollen-grains
entered.
DEVELOPMENT OF THE POLLEN-TUBE.
THE FIRST PERIOD OF GROWTH.
Germination of the Pollen-grain. — Germination of the pollen-
grain follows immediately after pollination. Ovules of Pinus
Strobus that were fixed on June 6, 1898, had not been pollinated,
but on June 13 pollination had occurred and the pollen-tubes
had been emitted ; similar evidence could be given for the other
species studied, but exact data on this point are at hand for Pinus
rigida only. Dispersion of the pollen occurred in this species
in the vicinity of Wellesley College in 1902 on May 27, and in
material fixed two days later, May 29, the first stages of germi-
nation are clearly evident. It is probable that the time is not
longer in the other species. This confirms Strasburger's ('92)
statement that germination takes place in Pinus at once after
pollination. Hofmeister ('51) was doubtless unable to detect the
early stages in the germination and hence was led to the con-
clusion that pollination and germination were separated by
several weeks in the Abielinea.
The pollen-grain increases slightly in size, the ventral or
concave portion of the wall becomes convex, then bulges out,
the exospore is ruptured, and the endospore is gradually pro-
longed into a tube. Immediately upon the formation of the
48 MARGARET C. FERGUSON
pollen-tube the tube-nucleus, as shown by Strasburger ('92)
moves away from the antheridial cell and into the pollen-tube
(figs. 75, 76, plate VII). According to Coulter and Chamber-
lain ('01, page 92), the tube-nucleus does not enter the tube
until the following April. That the tube-nucleus should at once
loose its association with the antheridial cell and accompany the
growing point of the pollen-tube is exactly what we should
expect from what we know, through the investigations of
Haberlandt ('87) and others, regarding the relation of the
nucleus to growth ; and, also, judging from the standpoint of
analogy, from the remarkable migrations of the tube-nucleus in
order to be near the growing point of the pollen-tube in Cycas
(Ikeno '98) and in Zamia (Webber '01).
Division of the Antheridial Cell. — Strasburger ('92) described
the antheridial cell in Pinus sylvestris as remaining unchanged
until the archegonia are formed in the following spring. Dixon
states that it divides about a month before fertilization, but from
a careful reading of the text one is given the impression that this
was an inference on his part rather than a demonstrated fact, as
he did not study material that was preserved earlier than April
24 and did not find the karyokinetic figure for this division.
And, in so far as I am aware, this mitosis has not been observed
in Pinus. Strasburger describes and figures it in Picea while
the pollen-grain is still within the anther.1
I have found great variation in the time at which the anther-
idial cell divides, not only in different species but in the same
species. It is rather interesting that Pinus Strobus, which
invariably lags somewhat behind the other species in all
other developmental phases studied, is remarkably precocious
as regards this step. Figs. 78, 80, and 81 were all taken
from material of Pinus Strobus which was collected and pre-
served on August 4, 1898, barely two months after pollina-
tion. In the same material, other pollen-grains were observed
in which the division of the antheridial cell had not yet taken
place ; but in material fixed somewhat later it was rarely found
undivided. The division of this cell has not been observed in
Pinus austriaca, but two cells have been found in the pollen-
grain in the middle of November and in February, and in such
1 See note at close of appendix.
LIFE HISTORY OF PINUS 49
instances the tube-nucleus can invariably be detected in the
pollen-tube. As pollen-grains containing but one cell were
also observed in this species on these dates, it might be
suggested that in the case of two cells the second prothal-
lial cell had persisted. The two cells, however, are exactly
similar to the stalk and the generative cell in their young con-
dition, and I see no reason for considering that they are not
these cells. On and after March 8 the antheridial cell of P.
austriaca is almost never found undivided. This date is given
for 1899 ; it would probably fluctuate in different years. Fig.
79 showNs the prophase of this division in Pinus rigida. Mi-
totic figures for this species have been found from April 21 to
May 13 of the same season. The division of the antheridial
cell in Pinus resinosa has been observed but once, this division
occurring on April u. All that can be said at present regard-
ing this mitosis in Pinus montana var. uncinata is that the gener-
ative cell and the stalk-cell are found as early as April 9. When
they are formed has not been determined.
In one preparation of Pinus Strobus two of the three pollen-
tubes which have almost reached the prothallium are furnished
with sperm- and stalk-cells, while in the third only the tube-
nucleus is found. On the apex of the nucellus there is a
pollen-grain which at this late date contains one cell, the
antheridial cell, still undivided (fig. 73). The nucleus of this
pollen-grain (fig. 74) is large, plump, and to all appearances
perfectly normal, and it is possible, though scarcely probable,
that it might still have divided. That one cannot 'trace a defi-
nite connection between the pollen-tube containing only the
tube-nucleus and this pollen-grain signifies little, for those who
have studied the pollen-tube of Pinus know that it is the excep-
tion rather than the rule when a given pollen-tube can be traced
through the lacerated dead tissue of the upper portion of the
nucellus to the pollen-grain from which it proceeded. Such a
condition as that described is rarely met with at so late a date ;
but occasionally during the summer and fall pollen-grains of
Pinus Strobus are found in which no cell-division has taken
place since pollination, although in the great majority of cases
50 MARGARET C. FERGUSON
the stalk- and the generative cell have been formed before the
middle of August.
These observations indicate that, while the division of the
antheridial cell takes place comparatively soon after the pollen-
grain has germinated in Pinus Strobus, and in some instances,
at least, before the winter's rest in P. austriaca, it is deferred
until the following spring in Pinus rigida and P. resinosa.
Furthermore, the time during which this cell may divide in a
given species may extend over several weeks, and in some cases
the division may never take place at all.
The Winter Condition. — A vertical section of an ovule of
Pinus Strobus collected on January 4 is represented in fig. 70,
plate VI. The spongy tissue surrounds a cavity crossed by
irregular strands of cytoplasm in which the free nuclei of the
prothallium are imbedded. In this instance the prothallium has
doubtless been displaced during fixation as it consists, normally,
at this stage, of a uniform layer of cytoplasm surrounding the
gametophytic vacuole and containing several nuclei. The
stalk- and the generative cell are enclosed within the pollen-
grain, and the tube-nucleus is near the apex of the irregularly
branched pollen-tube. This pollen-tube is shown more highly
magnified in fig. 83, plate VIII. At this time the pollen-tubes
have penetrated the nucellus almost to the point at which it joins
the free limb of the integument. The greatest depth to which
the tubes may have grown is not indicated in the illustration, but
this section was figured because it shows more clearly than any
other section in the series the cells of the pollen-grain and
the tube-nucleus. Other sections of the same ovule would have
shown pollen-tubes which had pierced to a greater depth into the
nucellus. The conditions of development as figured for Janu-
ary coincide perfectly with those which exist during the latter
part of October.
THE SECOND PERIOD OF GROWTH.
Renewed Activities in the Macrosporangium. — Growth is
very slow during the first period of development following pol-
lination, but with the renewed activities of spring the ovule
increases rapidly in size ; the central cavity of the nucellus
LIFE HISTORY OF PINUS 5 1
becomes greatly enlarged and is lined with the growing endo-
sperm. The cells of the nucellar cap which are penetrated by
the pollen-tubes during the previous season do not again become
active, but remain as deeply staining, thick-walled, dead cells.
The cells just beneath them, however, multiply rapidly, and
become literally packed with large starch-grains. A few of
the cells from this portion of the nucellar cap represented in
fig. 73, plate VII, are shown more highly magnified in fig. 89,
plate VIII. By the growth and increase of these cells, the dead
top of the nucellus with its pollen-tubes is lifted far above the
developing endosperm, so that the pollen-tubes, once so near
their goal, are now removed from it by a considerable distance
(figs. 70-72, plate VI).
Renewed Activities in the Male Gametophyte. — During the
rapid development of the ovule in the spring, the pollen-tube"*
increases little, if at all, in length, renewed activities in the male
gametophyte being first indicated by a further development of*
the cells within the pollen-grain.
The stalk-cell increases in size and its cytoplasm assumes a
vacuolate character. The growth of the generative cell is still
more marked, and its cytoplasm on the contrary becomes dense
and deeply staining. (Compare fig. 83, January 4, with fig.
84, May 3, plate VIII.) In Pinus sylvestris, as studied by Dixon
('94) and confirmed by Coulter ('97) in Pinus Laricio, the gen-
erative cell divides while it is within the pollen-grain. In the
species of pines which I have investigated, this division does
not occur until the generative and the stalk-cell have entered
the pollen-tube and the stalk-cell has passed below the gen-
erative cell. As the generative cell increases in size it stretches
out towards and into the neck of the pollen-tube, drawing after
it the stalk-cell, or possibly being forced out by that cell, the
two passing into the tube together.
Dixon states that only the naked nucleus of the stalk-cell
enters the pollen-tube, and in so far as I am aware, no writer
has described the entrance of the entire stalk-cell into the pollen-
tube in Pinus. The material which I have studied shows con-
clusively that the nucleus does not " slip out " of its cytoplasm
(figs. 83-86). The entire cell can be identified in the tube and
52 MARGARET C. FERGUSON
later in the egg. During the time that this cell is moving over
the generative cell its cytoplasm cannot always be differentiated
from that of the latter ; but when once the stalk-cell has passed
the generative cell, its nucleus surrounded by a sphere of very
vacuolate cytoplasm, scarcely more than a peripheral layer, is
again distinctly demonstrated (figs. 90 and 91). After pass-
ing the generative nucleus, the stalk-cell ordinarily takes up
a position between the generative cell and the tube-nucleus
(fig. 92), but occasionally it may pass the tube-nucleus (fig.
93). This phenomenon is always accompanied by a great in-
crease in the starch content of the pollen-tube, the tube being
in some instances almost filled with starch in the region of the
generative cell (fig. 91).
When the generative cell leaves the pollen-grain, its nucleus
is situated near the top of the cell, but the nucleus of this cell
evidently moves faster than its cytoplasm, and at the time when
the stalk-cell is passing over the generative nucleus this nucleus
has come to lie at or below the center of its cell (fig. 84, 90
and 91). Shortly after this the generative nucleus is again
observed at the uppermost part of its cytoplasm.
During its passage into the tube, the generative cell increases
much in size ; it has no definite cell-wall, and its cytoplasm forms
a large, irregular tongue about the nucleus. This cytoplasm in
no way suggests the alveolar structure of Butschli ('94) but is
distinctly reticular, differing in appearance from the nuclear
net only by its greater delicacy. This is shown more clearly
at a somewhat later stage.
The tube- and generative nuclei are now very similar in
structure, though each is sufficiently characteristic to be readily
recognized by one who is familiar with them. The tube-nucleus
has one large, usually homogeneously staining nucleolus, rarely
one or more smaller nucleoli, and it is furnished with a rather
scanty, delicate reticulum which is apparently poor in chromatin.
Either it is in a state of partial collapse, or, what is more prob-
able, it is very hard to fix at this period in its history, for its
outline is, as a rule, quite irregular at this time. The genera-
tive nucleus has one large, hollow or vacuolate nucleolus, and
commonly two smaller ones; its reticulum, though more abun-
LIFE HISTORY OF PINUS 53
dant than that of the tube-nucleus, is still delicate and often
shows a weak reaction to nuclear stains. The stalk-nucleus
has a very decided individuality which it maintains throughout
its entire history. It bears a strong resemblance from the first
to the nuclei of the nucellar tissue ; rarely, if ever, contains a
true nucleolus ; and its close-meshed reticulum is conspicuous
for its comparatively large net-knots or karyosomes.
Division of^the Generative Nucleus. — Comparatively few
students have occupied themselves with the growth of the pol-
len-tube in the Abietinea, and no one, in so far as I have been
able to determine, has described the cytological features attend-
ing the formation of the sperm-nuclei in this group.
Dixon ('94) describes this division in Pinus sylvestris as tak-
ing place about a month before fertilization , while the genera-
tive cell is still within the -pollen-grain; and Coulter ('97)
states, as already mentioned, that in his study of Pinus Laricio
he has been able to confirm Dixon's observations in the minutest
detail. At this time, as pointed out by Dixon, the nuclear and
cytological phenomena are very greatly obscured by the pres-
ence in the pollen-tube of large quantities of starch (fig. 91).
The starch, which resists the microtome knife and is therefore
easily displaced by it, not infrequently falls out and carries
away with it the free cells of the pollen-tube. The dead, deeply
staining tissue of the nucellus, representing that portion of the
nucellar cap which, was penetrated by the pollen-tubes during
the previous season, and in which the generative nucleus divides
(fig. 72, plate VI) is also very troublesome. Furthermore the
dense cytoplasm of the generative cell has a great affinity for
stains, so that when the archegonia and other portions of the
ovule are well stained, this cell often appears merely as a deeply
stained mass showing no differentiation of parts. Considering
the fact that I was led not only to expect this division to
take place within the pollen-grain but to search for it
some weeks earlier than it actually occurs in the species
of pines studied, together with the difficulties of staining, it is
not surprising that seven hundred slides of serial sections were
made, which means that more than two thousand pollen-tubes
were studied, before any definite clue was obtained as to the
54 MARGARET C. FERGUSON
true sequence of events in the development of the pollen-tube.
When once the mitotic figure was observed in the -pollen-tube^
scarcely more than a week before fertilization , and the fact
noted that special staining was necessary in order to study this
mitosis satisfactorily, further research was prosecuted with
comparative ease. I find no authority in Dixon's paper for the
statement recently made by Coulter and Chamberlain ('01)
which reads as follows: "The liberation and descent of the
body cell into the tube," etc., " has recently been described in
detail by Dixon." What Dixon ('94) does affirm is this : "Very
shortly after this it is found that the body-cell has broken free
from the stalk-cell and has divided into two cells, which are
almost equal in size. These cells are the male sexual cells.
During this process the wall of the stalk-cell is ruptured and its
nucleus follows the two cells resulting from the division of the
body-cell which move into the pollen-tube." And throughout
Dixon's paper there is no sentence that could be interpreted as
implying that the body-cell ever passes into the pollen-tube
before dividing to form the male sexual cells.
After the generative cell has passed into the pollen-tube but
while it is still in the upper dead portion of the nucellus, it gives
rise to the sperm-nuclei by a division which presents some new
and interesting features, although it resembles to a greater or
less degree certain mitoses described by various cytologists1
during the past few years.
When the generative nucleus has again come to lie in the
extreme upper portion of its cell, certain changes in the cyto-
plasm indicate that division is being initiated. At some little
distance below the nucleus the cytoplasm shows a finely granu-
lar structure which is not at this stage dense nor deeply stain-
ing. From this region irregular granular threads arise which
extend outward towards the periphery of the cell, those extend-
1 Of the long list that might be mentioned I have noted only the following :
Rosen ('95) in the root-tip of hj-acinth ; Osterhout ('97) in Equisetum" Swingle
('97) in SphacelartacccE ; Schaffner ('98) in root-tip of Allium Cepa ; Mottier
('98) in the embryo-sac of Liliumj Fulmer ('98) in pine seedlings: Hof ('98)
in Ephedra and other plants; Nawaschin ('99") in Plasmodiophora ; Nemec ('98
and '99) in various plants; Strasburger ('oo) in Vicia Faba ; Mottier ('oo) in
Dictyota; and Murrill ('oo) in Tsuga. Of animal cytologists I mention but
one, Hertwig, R. ('98) in A ctinospk cerium.
LIFE HISTORY OF PINUS 55
ing in the direction of the nucleus forming a hollow cone over
its lower portion (fig. 94, plate VIII). Gradually the granular
area increases in density and in staining capacity, at the same
time drawing nearer to the nucleus which is separated from it
by a hyaline court. Into this court delicate granular threads pass
(fig. 95, plate IX). When these threads reach the nuclear mem-
brane, the nucleus is forced so closely against the peripheral
layer of cytoplasm that its wall is frequently indented on the
upper side, while the condensation from which the so-called
kinoplasmic threads arise withdraws, or is forced by the growth
of the threads, further from the nucleus. A great number of
delicate anastomosing threads now extend, in the form of a
solid cone, from a point within the granular condensation up
towards and against the nucleus. The outer threads of the
cone pass over the lower portion of the nucleus and appear in
sections of the cell as closely packed against either side of the
nucleus. At the same time the entire cytoplasmic reticulum has
assumed a more or less radial arrangement about the condensed
area in which the spindle-fibers arose and from which some of
the more delicate threads extend into the surrounding cytoplasm
(fig. 96).
Coordinately with these changes in the cytoplasm, the chro-
matin of the nuclear net collects in spherical or irregular masses
on the reticulum, and sooner or later gives rise to a broad spi-
reme, along which the chromatic disks are distributed at regu-
lar intervals (figs. 94-98). After the segregation of the chro-
matin, there remains a delicate achromatic reticulum distributed
throughout the nucleus. This reticulum is also granular like
the chromatic network, but whether or not these granules rep-
resent the oxychromatin-granules of Heidenhain ('93 and '94)
I am unable to say. Webber ('01) has recently described and
figured a similar achromatic network in the generative cell in
Zamia. Whether the formation of the spireme precedes or fol-
lows the penetration into the nuclear cavity of the achromatic
threads seems to depend upon the length to which these threads
attain. They may become very long when their entrance into
the nucleus is delayed ; but more frequently a portion of the
nuclear membrane gives way, and some of the achromatic
56 MARGARET C. FERGUSON
fibers pass into the nuclear cavity before the spireme is estab-
lished (fig. 100). Rarely, the nuclear membrane appears pushed
in irregularly along its entire lower margin, as indicated
in figs. 96 and 98 ; as a rule, however, there seems to be one
deep, sharp indentation along one side of which the nuclear wall
first gives way (figs. 99 and 100). With the initial steps in the
disappearance of the nuclear membrane the nucleolus is either
not apparent or, if still demonstrable, it stains but feebly.
When the membrane disappears along the entire lower portion
of the nucleus, the kinoplasmic threads press so closely against
it that it can not be definitely demonstrated whether it passes
into the cytoplasmic and the nuclear reticulum or becomes fib-
rous and contributes to the formation of the achromatic threads
(figs. 101 and 102). The threads which have been packed so
closely against the wall of the nucleus now press into the
nuclear cavity and mingle with those which have entered from
below. And the dense, granular, cytoplasmic area from which
the threads diverge is gradually dissipated (fig. 103).
With the - disappearance of the wall along the lower part of
the nucleus, the achromatic nuclear network seems to undergo
a partial rearrangement. A portion of it is resolved into granu-
lar threads of more or less regularity which, in general, assume
a position parallel to the threads entering the nuclear cavity ;
some of them become attached directly to the ends of these
fibers, lose their granular appearance and doubtless contribute
to the growth of the elongating spindle-threads.
As the spindle-fibers proceed in their development across the
nucleus the chromatic spireme collects in the region of the future
equatorial plate, and becomes more or less massed together.
At the same time it assumes an homogeneous aspect and gives
rise by segmentation to the chromosomes (figs. 101-104). Some
of the ingrowing spindle-threads may extend across the nucleus
to the nuclear membrane, which is still present on the upper
side of the nucleus, but by far the greater number unite some
distance below this membrane to form several poles, thus giving
rise to a diarch spindle which, like the karyokinetic figures
occurring during the development of the pollen-grain is multi-
polar at its upper extremity and unipolar, or nearly so, at its
LIFE HISTORY OF PINUS 57
lower end. Gradually the poles of the upper portion draw
together, while the spindle is somewhat shortened oy the lower
extremity of the threads being again resolved into granules.
Finally a true bipolar diarch spindle is formed with the V-shaped
chromosomes oriented at the equatorial plate. Each pole termi-
nates in a slight granular condensation. The upper pole has
never been observed to reach the nuclear membrane, but fre-
quently coarse granular threads extend from the pole to the
membrane of the nucleus, and apparently act as supports for
the upper pole (fig. 105, plate X). These are evidently formed
by a rearrangement of the linin reticulum. The nuclear mem-
brane persists along the upper side of the nucleus until the late
telophase of the division (figs. 101—103, plate IX, and 104-107,
plate X).
As the chromosomes pass to the poles the central spindle
elongates, so that the daughter-nuclei are separated, as a rule,
by a greater distance than the length of the original spindle.
While this is characteristic of cell-division in general, it is occa-
sionally much exaggerated here, the daughter-nuclei being
apparently forced apart with considerable energy. The nucleus
which occupies the position nearest to the micropylar end of the
ovule often shows a deep indentation along its upper surface
as if a resistance had been met with in the peripheral layer of
cytoplasm (figs, in, plate X, and 113, plate XI). Not infre-
quently the upper nucleus is found almost entirely separated
from the cytoplasm (fig. 112). This, however, maybe due to
mechanical rupture during sectioning and staining. No cell-wall
is ever formed, and in only one instance was a condensation of
the spindle-threads in the region of the cell-plate observed (fig.
no). The spindle may contract at or near its center during
its dissolution, thus presenting the appearance of an hour-glass,
or it may give rise to such a condition as that shown in fig.
113. These appearances, with various modifications, are not
uncommon in this mitosis in Pinus. Hertwig ('98) describes
and figures a very similar lengthening of the spindle-fibers in
Actinospkarium. He also finds that the elongating spindle
finally bends along its median line so that the daughter-nuclei
come to lie near together in very much the same way as that
Proc. Wash. Acad. Sci., July, 1904.
58 MARGARET C. FERGUSON
shown in fig. 113. I am unable to trace definitely the origin of
this figure, but it is not improbable that it is caused by a con-
traction of the cytoplasm resulting from the cessation of the
force which effected the separation of the daughter-nuclei ; or
it may be produced by the resistance which the peripheral layer
of cytoplasm, along the outer surface of the upper nucleus,
offers to the growing fibers, thereby forcing them back upon
themselves as shown in the figure. When all traces of the
spindle have disappeared, the two sperm-nuclei are surrounded
by a common mass of cytoplasm, and there is never throughout
the later history of this cell the least suggestion of a dividing
wall.
The mitosis just described seems to be unique as regards the
origin and development of the achromatic spindle. Hertwig's
('98) fig. 3, plate V, illustrating an early stage in the division
to form the first polar body in Actmospkarium, bears a striking
resemblance to the prophase of this mitosis as illustrated in fig.
95, plate IX, of this paper ; but the origin of the figure shown by
Hertwig, and the later history of the division are very dissimilar
to that of the karyokinesis under consideration. The most
exaggerated instances of asymmetry in spindle-formation which
I have found recorded as occuring in plants is that described
and figured by Nemec ('992) in Solanum tuberosum, and more
recently by Murrill ('oo) in the division of the central cell in
Tsuga. In both these instances the nucleus lies at one side of
the cell, and the spindle-fibers are very much more prominent
on the free side of the nucleus than on the side adjacent to the
cell-wall. In another paper Nemec ('993) shows by experimenta-
tion that the form of the figure which gives rise to the extra-
nuclear spindle depends upon external forces or conditions. In
obedience to the law established by Haberlandt ('87) we should
expect to find the generative nucleus in that part of its cell which
is nearest the growing point of the pollen-tube, rather than at
the end more remote from it, and it may be that its passage from
the lower to the upper side of the cell is due to the fact that the
forces, instrumental in effecting the division, first become active
at a point below the nucleus, and exert a repelling action on it.
But I have at present no adequate explanation or theory to offer
LIFE HISTORY OF PINUS 5p
regarding the position of this nucleus at the time of its division.
Whether it is due to the origin of the karyokinetic figure, or
whether the unusual method of division is attributable to the
very eccentric position of the nucleus, I have not been able to
determine. It is evident, however, that the position of the
generative nucleus at the time of its division is such that the
spindle if extranuclear in origin must of necessity be unipolar,
since there is no cytoplasm, or almost none, above the nucleus
from which fibers could arise.
The blending of the linin reticulum with the cytoplasmic
network after the disappearance of the lower portion of the
nuclear membrane, and the relation of certain portions of the
achromatic nuclear reticulum to the ingrowing fibers are such
as to suggest an intimate relation between these structures.
That the spindle-fibers which originate in the cytoplasm and
apparently grow by a differentiation of its network are later fed
by the linin of the achromatic nuclear reticulum, there seems
little room for doubt. In fact, all the phenomena connected
with this division indicate that we are dealing, not with per-
sistent cell-constituents, but with different manifestations of one
and the same thing. In a word, we find no evidence here of
the presence in the cell of a definite kinoplasmic substance. I
am aware that these observations are directly opposed to the
views of the students of the Bonn laboratory, and many others
of the highest authority ; but the relations of nucleus, spindle,
and cytoplasm, not only in this division but in those to be
described in connection with fertilization, are such, it seems to
me, as to render no other conclusion in the case of these divis-
ions in Pinus possible. In 1895 Farmer arrived at a similar
decision regarding the origin of the spindle in spore-formation
in the Hepaticce, and Farmer and Williams ('98) in a study
of Fucus " do not regard the kinoplasm as a persistent proto-
plasmic structure, but as forming the visible expression of a
certain phase of protoplasmic activity." Hertwig ('98) expresses
himself as opposed to the view of a special spindle-forming sub-
stance in the protoplasm, while Wilson ('99 and 'oo) states that
the astral rays " grow by a progressive differentiation out of
the general cytoplasmic meshwork," and he finds in the echino-
OF TH
UN1VER'
6O MARGARET C. FERGUSON
derm's egg " no ground for a specific kinoplasm." The term,
however, is a convenient one and maybe employed consistently,
as suggested by Mottier ('oo), by those who do not find in kino-
plasm a morphological constituent of the cell, as descriptive of
that portion or manifestation of the protoplasm which is active
in spindle-formation.
Nothing has been said regarding the nature of the granular,
cytoplasmic condensation from which the achromatic spindle
takes its origin. It never has a definite boundary, though it is
often very clearly differentiated by its dense granular appear-
ance and its strong affinity for stains ; but at certain stages in the
division it may be inconspicuous or fail entirely of demonstration.
Such a vast amount of literature has accumulated during the past
decade regarding the nature and existence of the centrosome and
the centrosphere that one feels inclnied to avoid the subject alto-
gether. Yet the question may very properly be asked : Is this
condensation which forms the center of a system of radiating
fibers a centrosphere? It certainly is as clearly an attraction-
sphere as some bodies which have been described as such ; but
if we accept Wilson's ('oo) definition of the centrosphere, the
body under consideration cannot be so denominated, as no cen-
trosome has been observed at its center. More deeply staining
granules may sometimes be present within the condensation,
but these are not considered of any special significance as such
granules may be found anywhere in the cytoplasm.
Karsten ('93) describes the nucleoli in Psilotum as passing
out of the nucleus and assuming the role of centrosomes, and
Strasburger ('oo) considers that the nucleoli not only contribute
material for the formation of kinoplasmic threads, but that they
also make active the spindle-forming substance in the cytoplasm
— in other words, they act as the kinetic centers of the cell.
There seems to be no evidence that such is the case here, for
the nucleoli, after the condensation has arisen and the spindle-
threads have attained considerable length, are morphologically
the same as they were before the inception of the spindle.
Nemec (*99!) remarks that in the higher plants, where the cen-
trosome is not demonstrably present, the entire nucleus may
exercise the function of the centrosome. The idea of a diffused
LIFE HISTORY OF PINUS 6l
centrosome in the cells of the higher plants was suggested by
Guignard in 1897 and was again hinted at by Le Dantec in
1899. If we may accept Guignard's suggestion, then the kinetic
center of the cell in the higher plants is no longer indicated by
the presence of a definite organ, the centrosome, but the power
of this organ has become dissipated throughout the entire cell.
When that phase of cell-activity which has to do with spindle-
formation comes into play, the points at which it is centered
would naturally be indicated by a greater accumulation of the
microsomes, and thus an aster of more or less definiteness would
be formed, as when the individualized centrosome is present.
In the division of the generative nucleus in JPinus, the position
of the nucleus is such that the energy active in spindle-forma-
tion must perforce, if external to the nucleus, be centered at
some point below it. Such a centering of the activity would
naturally result in an attraction-sphere of unusual prominence ;
and there would be no occasion for its division since there is not
sufficient space above the nucleus for the organization of kino-
plasmic threads.
When these studies were undertaken, it was thought that it
would be interesting to determine whether any suggestions or
remnants of a cilia-forming body (called blepharoplast by Webber
in Zamid) still persist in the Conifers. Somewhat later, after
the present research was begun, MacMillan ('98) pointed out
the desirability of such a study both in Coniferce and Gnetales.
I have seen no indication of a structure which might be regarded
as a reduced blepharoplast, or as suggestive of a cilia-forming
body of any sort in connection with the formation of the sperm-
nuclei in Ptnus. Inasmuch as spermatozoids do not exist here,
such an organ, if present, must be functionless. But the cyto-
plasmic radiations which accompany the division of the genera-
tive nucleus in its early stages seem to differ in degree only from
those found by Webber ('97) in the generative cell of Zamia.
If we compare figs. 3 and 5 of Webber's paper with figs. 96 and
97, plate IX, of this paper, the question may be raised whether
in this cytoplasmic figure we may not have still persisting in
the cell the last vestiges of such an organ as that described by
Webber.
62 MARGARET C. FERGUSON
The endosperm has become a solid mass of tissue at the time
when the generative nucleus divides. The archegonia are still
comparatively small and quite vacuolate and the central cell has
not yet divided (fig. 72, plate VI).
Growth of the Sperm-nuclei. — After the mitotic figure has
entirely disappeared, the sperm-nuclei are separated by a con-
siderable distance. The form assumed by the cytoplasm sur-
rounding them seems to vary with the shape of the pollen-tube.
Gradually the two nuclei approach each other until they come
to lie in the extreme uppermost part of their cytoplasm (figs. 112,
plate X, 117, 118, plate XI). There is now considerable differ-
ence in their size. This inequality in size could be detected as
far back as the formation of the daughter-nuclei (figs. 109, no,
plate X). Belajeff ('91) was the first to figure and describe bi-
nucleated sperm-cells in the Gymnosperms. Coulter and Cham-
berlain ('oi), page 94, cite Belajeff as having observed an unequal
division of the generative cell in Taxus, the larger male cell func-
tioning, the smaller one remaining in the tube. But if I translate
the German correctly, what Belajeff says is that the nucleus of
the generative cell divides forming two nuclei which are about
one-half as large as the nucleus from which they were derived ;
one nucleus becomes larger and occupies a central position in
the plasma, the other nucleus is flattened and remains at the
periphery of the cell on its upper side ; the flattened nucleus
was never found surrounded by its own plasma, but in the same
plasma with the spherical nucleus. This is exactly the condi-
tion shown in Belajeff's figures, one of which is reproduced by
Coulter and Chamberlain. Jager ('99), however, has shown
two dissimilar sperm-cells in Taxus, the larger one in advance,
but he finds that occasionally the nucleus of the smaller cell
may exceed in volume that of the larger one. Jaccard ('94)
found two sperm-nuclei of the same size in Ephedra both sur-
rounded by the same mass of cytoplasm, and Coker (?O2) has re-
cently described the sperm-cell in Podocarpus as binucleated, the
smaller nucleus being above the larger and " thrust almost out
of the cell." No one, I believe, except the writer (i9Oiland2),
has recorded the presence of a single binucleated sperm-
cell in the Abietinece. In his earlier studies of the Gymno-
LIFE HISTORY OF PINUS 63
sperms, Strasburger ('69-92) was unable to demonstrate, satis-
factorily to himself, the character of the cells found in the
pollen-tube in Pmus, and he has not recently investigated the
male gametophyte in the Abietmece. Coulter ('97) described two
sperm-cells which were of the same size until within the arche-
gonium. Blackman ('98) stated that each sperm-nucleus was
clearly seen in the pollen-tube surrounded by its own cytoplasm,
but he did not figure them.1 Chamberlain ('99) figured the
sperm-nuclei, in Pinus Laricio, of equal size in the pollen-tube,
and showed them lying together in the cytoplasm of the tube.
Not having seen these cells within the archegonium before the
conjugation of the sexual nuclei, he accepted Coulter's state-
ment for the growth of one of them after their entrance into the
egg. According to Coulter ('oo) the " male cells in pines " are
alike in size. The same figures are reproduced by Coulter and
Chamberlain ('01).
As stated by the writer in 1901, two sperm-cells have not been
observed in any of the pines which I have studied ; but the
sperm-nuclei, which are of unequal size from a very early date,
remain, while in the pollen-tube, surrounded by a common cyto-
plasmic body (figs. 109-112, plate X; 113-118, plate XI, and
119-120, plate XII). As Strasburger ('92) observed, the larger
nucleus is always ahead, that is, on the side nearest the apex of
the pollen-tube. The smaller nucleus remains close against the
upper boundary of the cytoplasm, and suggests the condition in
Cycas (Ikeno '98) and Ginkgo (Hirase '98), where the stalk-
nucleus is forced entirely out of the cytoplasm surrounding the
generative nucleus. In the case of the smaller sperm-nucleus in
Pinus ) the action is not carried to so great an extent. Webber
('oi) has recently shown that such an interpretation as that re-
corded above for Cycas and Ginkgo is not true as regards the
stalk-nucleus in Zamia. One very interesting preparation which
I have obtained shows the smaller sperm-nucleus in advance of
the larger (fig. 114). Here it will be seen that the entire order
of arrangement has been changed, the stalk-cell and the tube-
nucleus being above the sperm-cell. But this abnormal arrange-
ment is only apparent, for it was found that the egg which had
1 See note at close of Appendix.
64 MARGARET C. FERGUSON
been approached by this pollen-tube had already been fertilized,
and the pollen-tube had turned aside and was passing up over
the top of the endosperm, as if seeking for another egg. The
position of the various elements of the pollen-tube is therefore
normal, the larger sperm-nucleus being in reality in advance of
the smaller. This suggests that, when a pollen-tube has con-
jugated with the egg, a substance may be secreted which repels
other pullen-tubes, as has been described in case of spermato-
zoids in the Bryophytes and Pteridophytes.
The formation of the sperm-nuclei shows most beautifully the
manner of the development of the nuclear reticulum. The
chromosomes unite end to end, giving rise to a homogeneous,
coiled band, before the nuclear membrane is formed. When
the nuclear-wall has been differentiated, the coil expands about
the periphery of the nucleus, while the band broadens, at the
same time becoming irregularly jagged along its margins.
These irregularities increase in length until finally those from
adjacent threads meet and fuse, thus giving rise to the reticulum
(figs. 107-110, plate X). When the sperm-nuclei have nearly
or quite come into contact they have as a rule reached their ma-
ture size. More than a year has now elapsed since pollination.
Elongation of the Pollen-tube. — Up to this time the pollen-
tube has elongated very slowly, having penetrated as yet little,
if any, beyond the nucellar tissue of the previous year's growth.
In this upper portion of the nucellar cap the tube may become
very broad, or it may branch freely (figs. 71, 72, plate VI, and
83, 87, plate VIII). When the sperm-nuclei have attained their
full size, the downward growth of the tube is exceedingly rapid,
travelling in from eight to ten days more than twice the distance
traversed during the entire preceding year. The path pursued
during this rapid growth is comparatively straight and the tube
is unbranched (fig. 73, plate VII). In Pinus Strobus, P. rigida
and P. austriaca about ten days intervene between the division
of the generative nucleus and fertilization ; in Pinus montana
uncinata, the two processes are separated by an even shorter
space of time.
The sperm-nuclei which at first present a very beautiful, rather
delicate reticulum (figs. 112, plate X, 117, plate XI), become
LIFE HISTORY OF PINUS 65
more dense as the pollen-tube advances through the nucellus.
Strasburger ('92) describes them as coarsely granular ; but, with
a high power, the presence of a reticulum which is sometimes
coarse and interrupted can invariably be made out in well pre-
pared material. By the time that these nuclei have reached in
their downward course the central portion of the nucellar cap
they have usually become very dense in structure (figs. 115 and
116), and frequently stain intensely, though they may show at
this time a weak reaction to dyes. The reticula of the two
nuclei may present the same appearance, or they may differ as
in the figures referred to above. The nucleolus, if it be present
at this time, is usually obscured by the dense network. Arnoldi
('oo) described the sperm-nuclei in Cefhalotaxus as being grad-
ually filled with metaplasm. I find no evidence of such a proc-
ess in the development of these nuclei in Pinus.
Archoplasmic areas similar to those figured by Chamberlain
('99) have been observed in connection with the sperm-nuclei,
but as such granular accumulations may occur at any point in
the cytoplasm of the sperm-cells no importance is attached to
them.
When the pollen-tube reaches the egg, its apex is abundantly
supplied with cytoplasm, in the upper part of which the tube-
nucleus lies. The sperm-cell is just above with the stalk-cell
still in contact with the lower portion of its cytoplasm (fig. 120,
plate XII). Still higher up the tube may contain many starch-
grains. There is never any doubt at this time as to the identity
of the stalk-cell and the tube-nucleus in the material which I
have studied. Yet Dixon ('94) states that they cannot be distin-
guished, and Coulter ('97) describes them as having lost their
original outline.
As many as six pollen-tubes have been found making their
way through the same nucellus, but, as a rule, not more than
three pollen-tubes renew their growth during the second season,
and frequently only two penetrate to the endosperm. The
effect of the pollen-tubes upon the upper part of the nucellar
tissue is very marked. The cells in the immediate vicinity of
the branched pollen-tubes early lose their protoplasmic contents
and their walls become crushed and broken. Those cells more
66 MARGARET C. FERGUSON
remote from the tubes do not suffer so severely, and retain their
protoplasm for a much longer time. Finally all the cells
representing the first year's growth of the nucellar tip loose
their content to a greater or less degree, and their cell-walls
become thickened and dead. During the rapid growth of the
pollen-tubes through that portion of the nucellar cap which
develops the second season, the effect of the tubes on the sur-
rounding tissue is less marked, though here, too, the cells with
which they come into contact are crushed and destroyed (fig.
73, plate VII). I have made no physiological investigations
regarding the action of these tubes on the tissue of the nucellus,
but, judging from the disappearance of the starch in the cells
just in advance of the tubes and the gradual disintegration of
those cells, it seems very probable that the destruction of tissue
is not due to mechanical reasons alone, but to the action of some
ferment or digestive substance as well. Various views have
been expressed concerning the action of the pollen-tube and the
directive agent in its growth by Molisch ('93), Miyoshi ('94),
Lidforss ('99) and others, but we are still far from a clear under-
standing as to the controlling factor in the movement. The
pollen-tube cannot be guided to the egg in Pinus by any peculiar
attraction existing between the sexual cells, for it grows with
normal rapidity when no sperm-cells are formed, and also when
the archegonia are in a state of disintegration.
SUMMARY.
Upon the germination of the microspore, three divisions fol-
low in rapid succession giving rise to the pollen-grain. At the
close of the prophase of each division the karyokinetic figure
is pointed at its lower extremity and very broad at the extremity
in contact with the dorsal side of the young pollen-grain. The
inner, incomplete, thick wall formed in the development of the
microspore persists as a part of the mature pollen-grain. It
probably serves as a strengthening layer, particularly at those
points at which the wall has been weakened by the expansion of
the exospore. When the telophase of the second division is
reached the first prothallial cell has become flattened against the
convex side of the spore-wall, its cytoplasm has been withdrawn,
LIFE HISTORY OF PINUS 67
and the nucleus has lost all signs of its former structure remain-
ing as a much flattened, deeply staining mass. At the close of
the third division, the second prothallial cell has suffered a simi-
lar fate . Both proth allial cells are furnished with cellulose-walls .
In the mature pollen-grain the prothallial cells are usually
represented by two broken, dark lines along the dorsal side of
the pollen-grain, but all vestiges of the first cell may have dis-
appeared. The antheridial cell projects from the convex side
of the spore at its middle point, and the tube-nucleus is always
directly below but in contact with the antheridial cell. Starch
is found in the pollen-grain at maturity and during its develop-
ment.
Pollination takes place between 42° and 43° north latitude
during the latter part of May or the first ten days in June. At
this time the macrospore-mother-cell is distinctly visible in the
center of the ovule, but slightly nearer its basal end.
In the young ovule the free portion of the integument, above
the tip of the nucellus, consists in cross-section of three layers
of cells. After pollination the arms of the integument become
erect, thus bringing the pollen-grains into the wide micropylar
canal. Then the inner layer of cells just above the pollen-
grains elongates rapidly, extending inwards and meeting at the
center. The pollen-grains having thus been made secure, the
elongated cells become divided into many small cells. It is
felt that the pit in the apex of the ovule in Pinus has been ex-
aggerated. There is rarely more than a slight concavity before
pollination. Through the action of the pollen-tubes it may be
somewhat deepened, but in normal conditions it does not become
" cup-like."
Two days after pollination, in Pinus rigida, the pollen-tubes
have been emitted. In the other species germination has been
shown to take place in less than a week after pollination, but
more exact data have not been obtained for these species. As
soon as the pollen-grain has germinated, the tube-nucleus severs
its connection with the antheridial cell and moves into the elon-
gating tube.
The division of the antheridial cell takes place in Pinus
Strobus during the first week in August. It sometimes divides
68 MARGARET C. FERGUSON
during the summer and fall in P. austriaca, but, as a rule, the
division takes place in this species very early in March. This
mitosis has been observed in P. resinosa during the second
week of April, and in P. rigida from the middle of April to
the middle of May. It is evident that this cell does not always
divide at a definite and fixed time, but that in a given species
the time during which it may divide extends over a considerable
period.
During the first season the pollen-tube grows very slowly,
and it may be broad and irregular in outline or it may branch
freely.
Shortly before fertilization the generative cell, followed by
the stalk-cell, moves into the pollen-tube. The stalk-cell soon
passes the generative cell and takes up a position near the tube-
nucleus. These changes and those immediately following are
frequently much obscured by the presence in the pollen-tube of
large quantities of starch.
When the macrosporangium enters upon the winter's rest, the
pollen-tubes have penetrated nearly to the line at which the in-
tegument becomes free from the nucellus and the tube-nucleus
maintains its position in the apex of the pollen-tube.
The generative cell is never limited by a well-defined cell-
wall, and consists at the time of its division of an irregular pro-
toplasmic body in the upper part of which the nucleus lies.
In the division of the generative nucleus the spindle is extra-
uuclear and unipolar in origin, a unique and heretofore unob-
served method of division.
The formation of the spindle indicates that the cytoplasmic
network and the nuclear reticulum have essentially the same
structure, and the spindle-fibers are apparently formed by a
transformation of both. The nuclear membrane persists along
the upper part of the nucleus until the early stages in the forma-
tion of the daughter-nuclei. This division takes place a little
more than a year after pollination and from a week to ten days
before fertilization, nearly thirteen months elapsing between pol-
lination and fertilization.
Two sperm-cells are never formed, but the sperm-nuclei
remain surrounded by a common mass of cytoplasm. An in-
LIFE HISTORY OF PINUS 69
equality in the size of these nuclei is very early apparent, and
becomes more pronounced as they reach maturity. The sperm-
nuclei soon come to lie together in the upper part of their cyto-
plasm and quickly attain their full size, the larger one being
invariably in advance. The nuclear reticulum, at first delicate,
soon becomes very dense, but there is no evidence of the pres-
ence in these nuclei of a special metaplasmic substance.
During the division of the generative nucleus the ovule in-
creases much in size, and the nucellar cap becomes several
times deeper than during the first season, thus carrying the
upper portion of the nucellus with its pollen-tubes far above the
endosperm.
At the time when the sperm-nuclei come into contact, or
nearly so, the pollen-tube has penetrated little, if at all, beyond
the nucellar tissue of the first year's growth. Now, however,
it again begins to elongate, and its downward course through
the new nucellar tissue is extremely rapid. The destruction of
the nucellar tissue through which the pollen-tubes travel, ap-
parently results not only from mechanical disturbances, but from
the entire dissolution of some of the cells through the action of
a ferment.
When just above the egg, the apex of the pollen-tube is filled
with cytoplasm. The tube-nucleus lies in the upper part of the
cytoplasm, and near it is seen the stalk-cell still in contact with
the lower portion of the cytoplasm which surrounds the sperm-
nuclei.
The existence of the diffused centrosome is suggested in con-
nection with the division of the generative nucleus, and there is
a possibility that, in the prominent cytoplasmic figure from
which the spindle takes its origin, we may have represented, in
its vestigial state, the cilia-forming body found in the lower
Gymnosperms.
7O MARGARET C. FERGUSON
CHAPTER III.
MACROSPOROGENESIS.
THE FEMALE CONE.
The Macrosporangium. — During this investigation I have
made no attempt to study the early development of the ovule
except to note definitely the date of its origin.- The pistillate
strobili cannot be detected in Pinus Strobus with the most careful
examination until the last of April or the first of May. In the
other species studied they are about one and one-half milli-
meters long at the middle of March, and it is possible that in
these species they were organized in the autumn, but I have not
been able to find any evidence that such is the case. I have
recently, November 25, 1902, attempted to discover the young
cones of Pinus rigida and P. austriaca, but, as formerly, the
search was futile. I was led to look again for these strobili in
the autumn by the recent statement of Coulter and Chamberlain
('01). On page 79 of their book on the morphology of the
Gymnosperms, I find this sentence, based on a study of Pinus
Laricio: " In June the archegonia are ready for fertilization,
which occurs about the first of July, at least twenty-one months
after the first organization of the ovule." This by a very simple
mathematical calculation places the " organization of the ovules "
on October i.
I have not only been unable to detect the pistillate cones
before the approach of winter, but in the tiny cones of Pinus
rigida^ P. auslriaca and P. montana uncinata, fixed on March
14 there is not the least suggestion of ovules, the entire
cone consisting in each case of a broad axis on the margin of
which are slight elevations or papillae — the beginnings of the
bracts which subtend the ovuliferous scales (fig. 121, plate
XII). The first indications of the ovules are found in these
species about the last of April or the first of May. In material
of Pinus Strobus fixed on May 31, 1898, tthe position of the
ovule can be detected only by a slight bulge on the inner sur-
face of the ovuliferous scale, the integument not yet having
been differentiated. One week later, June 6, the ovule is
••
\c
LIFE HISTORY OF PINUS 7 1
found fully organized and nearly ready for the reception of the
pollen-grains (figs. 122, and 123). The evidence is conclusive
that the ovules are not organized in the species of pines
studied by the writer until about three weeks or less before
pollination, and seven months later than in Pinus Laricio as
recorded by Coulter and Chamberlain. This is the more surpris-
ing when we -consider that P. austriaca is at least a variety of
P. Laricio , and, according to some authorities, it is a synonym
for that species.
It is not my purpose to enter into a discussion of the origin
and cellular development of the female cone, nor yet of the
homologies of its parts. These points have been fully investi-
gated by Celakovsky, who has frequently published papers on
this subject from 1879 to the present time, and the many theories
advanced by different writers regarding these structures have
recently been brought together and reviewed by Worsdell ('oo).
FORMATION OF THE AXIAL ROW.
The Macros-pore-mother-celL — The origin of the sporog-
enous tissue from a hypodermal cell or cells was described by
Strasburger for several Gymnosperms in 1879, an(* this idea
without further confirmation has come down to the present time.
While this may be true for many Gymnosperms, and possibly
for Pinus, I find no evidence, direct or indirect, that the macro-
spore-mother-cell is derived from a hypodermal cell in the pines
investigated. When the mother-cell is sufficiently differentiated
to be distinguishable from the other cells of the surrounding tissue,
it is found to lie deep within the nucellus ; and there are no rows
or axial strands of cells lying above it to suggest its derivation
from a hypodermal cell. On May 8, 1902, the ovules of Pinus
rigida were sufficiently developed to show clearly the separation
into nucellus and integument, and a like condition was found to
exist in P. Strobus on June 6, 1898. In both instances, so
far as one is capable of determining, every cell of the nu-
cellus is exactly like every other cell (fig. 123), and the
same condition obtains in the other species at this time. One
week later, as illustrated for Pinus rigida^ the macrospore-
mother-cell can first be distinguished, and the so-called spongy
72 MARGARET C. FERGUSON
tissue is clearly differentiated about it (fig. 124). The mother-
cell in this instance has relatively the same position in the ovule
as that shown in fig. 66, plate VI, which was taken from an
ovule collected twelve days later. If this cell be the direct de-
scendant of a hypodermal cell, it has now become deep-seated
by the addition of cells above it ; but there is nothing in the
arrangement of the cells of the nucellus either before the
appearance of the mother-cell or after it to denote such a course
of development.
The mother-cell is first detected by its larger size and by its
failure to stain as deeply as do the other cells of the nucellus.
In the first stages of growth the nucleus almost fills the cell
(fig. 125), and its weakened capacity for staining is doubtless
due to its rapid growth without a proportional increase in the
amount of nuclear substance. The nucleus contains in this
young stage a delicate reticulum with a varying number
of larger and smaller net-knots, and from two to four small
nucleoli, not differing materially, except in size and staining
power, from the nuclei of the adjacent tissue. This cell in-
creases considerably in size before its division so that it becomes
very conspicuous in the nucellus, its reticulum taking the chro-
matin-stains with greater avidity than at an earlier period.
The season of growth for the macrospore-mother-cell may
extend over about three weeks. The early stage shown in
figs. 124 and 125 represent its size on May 15, 1902, and the
spireme stage illustrated in fig. 126 indicates the condition of
this cell on June 5 of the same year.
First Division of the Macrospore-mother-cell. — After the
mother-cell has attained its full size, the reticulum of the
resting nucleus gradually becomes more open, the chromatic
granules become more prominent and there arises a beauti-
ful, regularly moniliformed, more or less interrupted skein,
but a true spireme is not formed until after synapsis (fig. 126).
This somewhat branched thread is very delicate, the chro-
matic discs are uniform in size and distributed upon the linin
with great regularity. It is probable that these apparently
homogeneous discs, which have doubtless been derived from
the fusion of the smaller chromatic granules, would, under
LIFE HISTORY OF PINUS 73
greater magnification, be resolved into slightly irregular and
roughened bodies, as in the prophase of the heterotypical mitosis
in the microspore-mother-cells, but with the powers of the micro-
scope at my command, I have no evidence that such is the case.
The phenomenon of synapsis is as marked here as in the
primary mitosis of the microspore-mother-cell, but the contracted
mass is less dense, probably because of the smaller size of the
nucleus and the consequent diminution in nuclear substance
(fig. 127, plate XIII). With the recovery from synapsis the linin
thread is seen to have increased in thickness, and the chromatin-
granules are irregularly distributed upon the continuous spireme,
which gradually comes to fill the entire nuclear cavity with
its open uninterrupted coils (figs. 128 and 129). The chro-
matic substance again collects into definite areas of varying
dimensions, which are united by clear portions of the linin-
band, and the longitudinal splitting now becomes apparent.
Condensation and segmentation follow, and the distinct chro-
mosomes, in the reduced number, become evident (figs. 130,
132 and 133). The forms of the chromosomes are similar to
those already described in connection with the division of the
microspore-mother-cell (figs. 132-136). Because of the com-
paratively small size of these nuclei, the steps by which the
irregularly shaped chromosomes are derived could not be traced
with the same degree of confidence as in the microspore-mother-
cell ; but the entire phenomenon is such as to indicate very con-
clusively that the process is practically the same in both.
The spindle, at first a multipolar diarch, early becomes bi-
polar and during metakinesis it is very sharply so. The poles
do not reach the walls of the cell, but a few threads sometimes
radiate from them and extend to the ectoplasm. There may
be a slight granular condensation in the neighborhood of the
poles but it is never prominent and often does not appear at all.
The chromatic segments become short and broad at the equa-
torial plate, and their separation into daughter-chromosomes
presents the figure characteristic of the heterotypic division.
Unsplit ends of the chromosomes extend outward in the plane
of the equatorial plate, thus giving rise to dark clumps of chro-
matic substance along the median line (figs. 134-137). The
Proc. Wash. Acad. Sci., July, 1904.
74 MARGARET C. FERGUSON
passage of the one-half chromosomes to the poles has not been
observed. Resting nuclei are formed during the telophase of
the mitosis, and a cross wall divides the mother-cell into two
compartments (fig. 138).
From the foregoing it is evident that the first division which
takes place in the macrospore-mother-cell is heterotypic in
nature, and agrees in all essentials with the primary mitosis
within the microspore-mother-cell. This is in accordance with
the conclusions reached by all other investigators who have
recently studied the tetrad divisions occurring within the ovules
of various Phanerogams.
Second Division of the Macrospore-mother-celL — Beginning
with the telophase of the first division considerable variation
may occur in the subsequent steps in the formation of the axial
row. A cell-plate is always formed between the daughter-
nuclei though it may remain very delicate, consisting of little
more than a condensation of the ectoplasm. The daughter-
cells may be very similar in appearance, excepting that the
lower one is usually the larger, and in such instances both nuclei
enter the resting stage, presenting a clear, definite reticulum
(figs. 138, and 141). More often, however, the lower cell is
much larger than the upper one and the nucleus of the upper
cell does not enter into the complete resting stage, but early
shows signs of disintegration. The chromosomes may unite to
form a spireme as usual, but development may then cease
without the organization of a network, and the diffuse reaction
of the nucleus to stains shows that disintegration has begun
(figs. 139, 140).
I have but a single preparation showing the second division
of the macrospore-mother-cell, and I can therefore offer no con-
clusions of any value regarding the nuclear phenomena accom-
panying the mitosis. From this figure it appears that the
spindle originates as a multipolar diarch as in the first division,
and both nuclei in this instance are dividing at the same time.
During the initiation of the spindle the chromosomes are short
and thick, somewhat irregular in outline, and apparently in the
forms of U's, V's and rings. The reduced number of chromo-
somes occurs in both of the dividing nuclei (fig. 142).
LIFE HISTORY OF PINUS 75
The state of disintegration referred to above is always con-
fined to the upper of the two daughter-cells and never occurs in
the lower one, except in those cases in which the whole ovule
is undergoing destruction. The lower cell invariably divides
again and the basal cell thus formed constitutes, in every instance
observed, the functional macrospore. The lack of constancy
in the division of the upper cell would naturally give rise to
some axial rows of four cells and some of three, and this is
exactly what we find (figs. 144, 145, plate XIV). Fig. 143 shows
the second division of the lower cell just completed, and it is evi-
dent from the structure and appearance of the uppermost nucleus
that it would never have divided. In the axial row presented in
fig. 144 some time has elapsed since the mitosis was completed,
as evidenced by the increase in size of the lowest cell of the row.
The upper of the two cells formed as a result of the first mitosis
still remains undivided, and, moreover, it would not have divided
later, judging both from its appearance and from the fact that the
rapid growth of the initial cell of the female gametophyte would
soon have been instrumental in effecting its obliteration. Juel
('oo) finds that these cells do not divide simultaneously in Larix^
but he does not find the division completed in the lower cell before
it begins in the upper one. In the single preparation showing
the second division in the macrospore-mother-cell, both nuclei
are dividing, and both are in the same stage of the prophase,
but this does not necessarily mean that when both cells divide
they always do so synchronously. This lack of uniformity in
the number of cells in the axial row is not peculiar to Pinus ;
it has been observed by many investigators in a large number
of plants including both Gymnosperms and Angiosperms.
Coulter and Chamberlain ('01) figure an axial row of four
cells in Pinus Laricio^ and, as above indicated, such an axial
row is frequently met with in the species of pines which I have
studied, but it is much more common in Pinus austriaca than
in the other species (figs. 145, plate XIV, 142, plate XIII, and
261 , plate XXIII) . There is no doubt whatever, after a study of
many preparations showing the axial row, that in the great
majority of cases in Pinus Strobus and P. rigida the upper cell
remains undivided and that the usual axial row in these species
76 MARGARET C. FERGUSON
consists of three cells. The axial row represented in fig. 144, for
instance, is a beautiful object, clearly and definitely differentiated
from the surrounding tissue, yet there is not the least ground
for supposing that the upper cell has ever divided. Such a
figure as this represents the characteristic axial row in Pinus
Strobus and P. rigida> while the axial row of four cells illus-
trated in fig. 145 is typical for P. austriaca. This point has
not been sufficiently studied in the two other species to admit of
generalizations for them. The axial row, then, varies from
three to four cells in the same species, but there is a tendency
in some species to form three and in others to form four cells.
Significance of the Tetrad Division Within the Ovule. — We
have observed that at a certain point in the development of the
ovule in Pinus a centrally located cell becomes differentiated
from those surrounding it by its greater size and the more
vacuolate character of its cytoplasm. This cell after under-
going a period of growth and rest gives rise to the reduced
number of chromosomes by a peculiar method of division known
as the heterotypical division, and this mitosis, as is characteris-
tic in spore formation, is quickly followed by a second division,
at least in the lower cell. The basal cell resulting from this
last division passes through a season of growth extending over
several weeks, as we shall shortly see, and finally, by repeated
divisions, gives rise to the female gametophyte. The process
of division is in all essentials exactly similar to that which takes
place within the microspore-mother-cell, and results, as there,
in spore-formation. Nuclear phenomena attending the early
development of the female gametophyte have not been carefully
investigated until comparatively recent times, but wherever
studied the conclusion has been unhesitatingly drawn that in
the ovule, as within the anther, a true spore-formation takes
place.
The essential character of a spore is, manifestly, not that it
should have a certain arrangement relative to its sisters within
the mother-wall, neither is the presence or absence of a wall of
vital importance to its existence unless, indeed, the spore is to
be disseminated. Rosenberg ('01) finds the pollen-grains to be
filiform in Zostera and arranged side by side ; Strasburger ('01)
LIFE HISTORY OF PINUS 77
and Gager ('02) show that the descendants of a pollen-mother-
cell in Ascleptas have a linear arrangement ; while Juel ('oo)
discovers that in the Cyperacece three young pollen-grains or
microspores abort and the fourth remains permanently within the
microspore-mother-wall. Yet from the standpoint of origin
alone, no one hesitates to call the young pollen-grains of these
plants microspores. Juel ('oo) affirms that the heterotypic divi-
sion must be the criterion by which we decide whether or.no we
have a true tetrad-division, and he concludes that in Larix the
embryo-sac-mother-cell is homologous with a spore or a micro-
spore-mother-cell. Schniewind-Thies ('01) reaches the same
conclusion for Angiosperms ; and Lloyd ('01) asserts that the
division of the embryo-sac-mother-cell in the Rubiacece is a
true tetrad-division, and the four resultant cells are spores.
Other instances where similar conclusions have been reached
might be cited, but the above is sufficient to demonstrate that
the most recent studies along this line point conclusively to a
normal spore-formation within the ovule, and do not confirm
Campbell's ('02) statement that a true tetrad-division is usually
absent in the ovule of spermatophytes.
For many years botanists have been involved in a contention
regarding the true nature of the embryo-sac in Phanerogams.
A paper was published by Atkinson in 1901 reviewing the
interpretations made by earlier writers and suggesting as a
solution of the difficulty that spores, no longer being necessary
in the higher plants, had dropped out of the cycle of develop-
ment in these plants. That is, the female gametophyte arises
in the higher plants without the intervention of spores. While
the results of recent investigations do not serve to strengthen
this view, the theory is a most interesting one and the paper
has further served an excellent end in stimulating thought and
research along this line. Mottier observed one instance in
which the first division of the embryo-sac-mother-cell was homo-
typic, or, if we use Strasburger's ('oo) term adopted through-
out this paper, typical, and the number of chromosomes was not
reduced. Juel found the same to be true normally in Anten-
naria alpina, a species of Antennaria in which the embryo
develops parthenogenically. In both instances we have an
78 MARGARET C. FERGUSON
illustration of development within the embryo-sac without the
intervention of a spore, but these are apparently isolated and
exceptional cases.
The whole difficulty seems to me to lie in the fact that all
along we have been endeavoring to make a morphological unit
out of that which is primarily a physiological unit, and not
necessarily a morphological one, although it may be so. It has
been shown conclusively that in Larix and Pinus among the
Gymnosperms a true macrospore is formed which germinates
within the macrosporangium and gives rise to the female gamet-
ophyte — both a morphological and a physiological unit. But
as we advance to the Angiosperms there is a shortening of on-
togeny in the female gametophyte, the most extreme case being
represented by Lilium. Mottier ('98) demonstrated the fact
that the division of the embryo-sac-mother-cell in Lilium is a
true tetrad division and we cannot, therefore, it seems to me,
escape the conclusion that the resultant four cells are spores.
But once rid ourselves of the idea descended from Hofmeister,
that the mother-cell of the embryo-sac is always a macrospore,
and the product of its development, therefore, always a single
gametophyte, and many difficulties vanish. Lloyd ('02), in his
recent discussion of this subject, accepts the heterotypical divi-
sion as the criterion for spore formation, and then explains the
condition in Lilium^ where the first four cells of the embryo-
sac are spores, by " regarding the gametophyte as an individ-
ual by coalescence" It appears to me not only more simple
but more plausible to consider that we have here four gameto-
phytes each reduced to two cells. The embryo-sac is still here
as elsewhere (with the exception of parthenogenic plants), a
physiological unit whose function is to give rise to a new plant
through the sexual process, but it is morphologically a complex
made up of several individuals. Whether all eight cells thus
formed are considered as potential eggs is immaterial, practi-
cally, but one retains the power to respond to the sperm-cell,
though the others have been shown to be capable of fertiliza-
tion in some instances. Ordinarly, however, they remain ster-
ile and have come to have a vegetative or nutritive function
only. All work together for one end and in that sense may
LIFE HISTORY OF PINUS
79
make " an individual by coalescence," that is, they are physi-
ologically one.
This is not the place to enter into a detailed discussion of the
homologies of the embryo-sac, but I believe that the suggestion
herein made will form an interesting working basis, and it may
bring us nearer to a true conception of these structures than we
have yet attained. But whatever our opinion regarding the ele-
ments within the embryo-sac, it is clear that we cannot longer
use the terms macrospore and embryo-sac interchangeably as so
many writers have done. We now know that a tetrad division
may occur within the ovule and it has been shown that the
embryo-sac may result from the germination of a single macro-
spore, that it may be formed directly from the macrospore-
mother-cell, or that it may have its origin in one of the daughter-
cells formed as the result of the heterotypical division. In any
case would it not be far less confusing if we should designate
the multicellular bodies, developed within the macrosporangium
and the microsporangium of the higher plants, as embryo-sac
and pollen-grain, or female and male gametophyte, respectively,
and should retain the terms macrospore and microspore for the
true spores in their one-celled stage?
LATER HISTORY OF THE AXIAL ROW.
The Fate of the Upper Cells. —Whether the number of cells
in the axial row of Pinus be three or four the female gameto-
phyte is always the product of the lowest cell. Very shortly
after the second division is completed, the upper cells of the
axial row give evidence of disintegration, while the basal cell
increases much in size, its nucleus becoming very large. The
nuclei of the four spores in Larix are very similar, Juel ('oo),
fig. 18, but in Pinus the basal cell is markedly different from
the others at a very early date (figs. 144, 145, plate XIV). The
upper cells of the axial row gradually disintegrate, and are
crowded to one side by the growth of the macrospore, remain-
ing for a time as deeply staining, amorphous masses which
finally disappear altogether (figs. 69, plate VI and 147, 148,
plate XIV). Instances in which one of the upper cells of the
axial row in Angiosperms becomes the functional macrospore
80 MARGARET C. FERGUSON
are not rare. Campbell ('oo) has recorded such a condition in
the Aracece, Lloyd ('01) in certain Rubiacea, and Karsten ('02)
in the Juglandacecz. But, so far as investigated, the sequence
of events following the establishment of the axial row in the
Abietinece. results in the obliteration of all but the lowest cell.
I have avoided using the term " potential macrospore " in con-
nection with the upper cells of the axial row, because the upper
of the two cells first formed does not always divide and in such
instances it cannot properly be designated as a spore since
development ceased before spore formation was completed.
Growth of the Macrospore. — Starch is sometimes found
within the cells of the axial row, though never in such abundance
as in the cells of the adjacent tissue (fig. 143). It may become
very abundant within the macrospore during its period of growth,
and is sometimes found pressed so closely against the nucleus
as to actually produce indentations in its membrane (fig. 146).
The reticulum of the nucleus of the functional spore is very
scanty during its growth period, but later it presents the appear-
ance of an ordinary resting nucleus. The cytoplasm, never
abundant, forms at an early date a loose, granular network.
Later the nucleus is connected with the ectoplasm by delicate
strands which are gradually withdrawn into the peripheral cyto-
plasm, until there is thus formed in the one-celled stage a
definite layer of cytoplasm lining the wall of the macrospore,
and inclosing a large central vacuole. The nucleus moves to
one side of the cell, usually the upper side, imbeds itself in the
cytoplasm and awaits further development (figs. 147, 148).
The organization at so early a period of this definite peripheral
layer of cytoplasm has not, I believe, been demonstrated for
any of the other Gymnosperms. Finding the cavity containing
the developing endosperm crossed by irregular strands of cyto-
plasm as illustrated in fig. 70, plate VI, I had the impression
for a long time after these studies were begun, as stated in an
earlier paper (ipoi3), that such a condition, as that described
above for the resting macrospore, did not obtain until the
beginning of the second period of growth. This layer of cyto-
plasm is very easily displaced by the action of the fixing fluid,
but with care it may be obtained in an apparently normal con-
LIFE HISTORY OF PINUS 8 1
dition. I now have an abundance of preparations which show
not only that the wall layer is instituted in the one-celled stage,
but that it persists as long as free cell-formation continues in the
endosperm. The only reference which I find regarding the
establishment of the wall-layer of cytoplasm in any of the
Gymnosperms is the following statement made by Coulter and
Chamberlain ('01), with reference to Pinus: " Probably when
but two or three free nuclei have appeared the nuclei become
imbedded in a parietal, cytoplasmic layer."
SUMMARY.
The female cones can be distinguished early in March,
excepting in Pinus Strobus where they do not appear until the
very last of April. The ovules cannot be detected until about
three weeks before pollination.
There is no evidence that the macrospore-mother-cell arises
from a hypodermal cell. When first differentiated it is cen-
trally placed nearer the chalazal end of the ovule.
The division of the macrospore-mother-cell is a true tetrad-
division and the cell which gives rise to the female gametophyte
is a true spore.
Of the two cells formed as a result of the heterotypic division
the lower one always divides again, the upper one may. An
axial row of three cells seems to be the rule in Pinus Strobus
and P. rigida, and one of four cells the rule in P. austriaca,
though neither is constant in any of the species. The lowest cell
of the axial row always becomes the functional macrospore.
The two or three upper cells of the axial row begin to disin-
tegrate very soon after they are formed and are finally absorbed
by the enlarging macrospore.
The lower cell passes through a long period of growth during
which the cytoplasm is withdrawn from the central portion of
the cell and forms a uniform layer lining the wall of the macro-
spore. The nucleus moves towards the upper side of the cell
and imbeds itself in the peripheral layer of cytoplasm.
The suggestion is made that the embryo-sac may or may not
be a morphological unit, but that it is essentially a physiological
unit, existing for the purpose of sexual reproduction. Such a
82 MARGARET C. FERGUSON
conception of the embryo-sac seems to the writer to form a more
satisfactory basis for a rational explanation of the structure, or
composition, and homologies of the embryo-sac than do any of
the existing theories regarding the nature of this body.
CHAPTER IV.
THE FEMALE GAMETOPHYTE.
DEVELOPMENT OF THE PROTHALLIUM.
The First Period of Growth. — We are indebted to Hof-
meister ('51) for our first definite knowledge regarding the life
history of the female gametophyte in the Gymnosperms. It is
true some errors in observations were made, but they were inter-
mingled with much that has stood the test of the most modern
research. In 1879 Strasburger declared the " transitory endo-
sperm " described by Hofmeister to be a fallacy, but he himself
fell into quite as grave an error, though in the opposite direction,
when he stated that the primary nucleus of the embryo-sac
remained undivided during the first year, an observation since
corrected by himself.
As already stated, the young macrospore immediately organ-
izes a peripheral layer of cytoplasm and passes through a period
of growth which continues for six weeks or more. The degree
of development which has been attained by P. austriaca
on June 13, 1898, is shown in figs. 145 and 147 ; the first
division of the macrospore-nucleus in this species occurred
on July 29 of the same year, as illustrated in figs. 149 and 150.
The germinating macrospore had now enlarged to such an
extent that it was found necessary to reduce the scale of mag-
nification at this point so that a comparison of the figures does
not present, visually, the amount of growth which ensues
between the organization of the macrospore and its first division.
Pinus differs substantially in respect to the very marked growth
of the macrospore before the first division of its nucleus from
Larix where two nuclei are formed before there is any con-
siderable increase in size of this cell (Juel ('oo) plate xv, figs.
LIFE HISTORY OF PINUS 83
18-20). The persistence of the potential megaspores in Larix
at this time is also in very striking contrast to Pinus, where the
other cells of the axial row have become entirely absorbed before
the germination of the macrospore occurs (figs. 147-149).
The third division of the macrospore-mother-cell, or the first
division of the macrospore-nucleus, takes place during the very
last of July or the first of August in all the species studied,
and is of the ordinary or typic method. It differs from the
mitoses occurring in the vegetative tissue of the sporophyte
only in presenting the one-half number of chromosomes (fig.
150). The daughter-nuclei may remain at one side of the pro-
thallial cavity, but more frequently they pass to opposite sides
as in the development of the embryo-sac in Angiosperms (fig.
151). The second mitosis follows rather quickly, and is already
completed in Pinus Strobus on August 4 (fig. 152). Nuclear
divisions follow until several free nuclei have been formed.
The observations of Strasburger ('79), and of all later students
of the Gymnosperms, upon the simultaneous division of the free
nuclei of the endosperm have been confirmed. On October
12, 1898, sixteen nuclei were observed in the cytoplasmic layer,
all being in the spireme stage of division. On October 15 of
the same year sixteen nuclei, all presenting the equatorial plate-
stage of mitosis were found in the cytoplasm of the prothallium,
(figs. 153-155). The karyokinetic figure is sharply bipolar,
each pole ends in a slight condensation of the cytoplasm, and
the chromosomes are clearly of the reduced number.
I find no evidence that any further divisions occur during
the first period of growth and it is probable that the thirty-two
nuclei which result from the division just described pass into
the resting stage and remain inactive during the winter. But I
have not examined a sufficiently large number of preparations
with this point in mind to affirm that the prothallium of Pinus
invariably enters upon its long period of rest in the thirty- two
nucleated stage. The number may not be fixed even in the
same species, but it is certain that it is never large. The pro-
thallium, therefore, at the close of its first season of growth is
a spherical body composed of an ectal layer of cytoplasm in
which are imbedded, in many instances at least, thirty-two free
84 MARGARET C. FERGUSON
nuclei. This thin cytoplasmic shell encloses a large central
vacuole which is reported by Strasburger, Arnoldi and others
to be filled with a fluid substance. I have made no observa-
tions regarding the cell-sap of this large vacuole and can neither
affirm nor deny its presence.
The Second Period of Growth. — It has been seen that the
ovular development in Pinus is very slow during the period imme-
diately subsequent to pollination, but with the renewal of growth
in the spring development becomes much more rapid. Coor-
dinately with the enlargement of the ovule already described,
the endosperm cavity increases in size until it occupies almost
the entire basal and central portions of the nucellus, presenting
in longitudinal section the figure of an ellipse (fig. 71, plate
VI). The thin peripheral layer of cytoplasm with its free nuclei
persists until the latter part of May, and free nuclear division
continues to take place within it until a large number of nuclei
are formed. Jager ('99) estimated that there are 256 free nuclei
formed in Taxus, and Hirase ('95) made the same observation
in Ginkgo. The number is certainly much larger in Pinus.
More than 500 free nuclei are present early in May and about
2,000 have been counted in Pinus Strobus at the time when the
nnclei are being separated by the development of dividing walls.
The free nuclei are considerably larger in surface view than
the nuclei of the nucellar tissue, but in side view they often
appear somewhat flattened. They have the structure of typical
resting nuclei (figs. 156-159, plate XV). Each contains, almost
invariably, two rather large nucleoli surrounded by clear areas.
The reticulum is close and studded with irregular granules, but
the net-knots are not so prominent as in the nuclei of the nucel-
lus. They simulate very closely the nuclei of the sheath-cells
at certain stages in the development of the archegonia. The
cytoplasm in surface view presents a pseudo-alveolar structure
consisting of a coarse, granular reticulum enclosing numerous
vacuoles (fig. 156). During the late telophase in the division of
the free nuclei of the prothallium the complicated karyokinetic
figure characteristic of free nuclear division becomes very con-
spicuous, and is evidently formed as a result of the rearrange-
ment of the cyto-reticulum (fig. 159).
LIFE HISTORY OF PINUS 85
At some time during the latter part of May in Pinus Strobus
and about the middle of the month in the other species free
nuclear division ceases and cell-walls are developed between the
nuclei. The development of the prothallium from this point
on was studied by Sokolowa ('80), and her observations have in
general been confirmed by all more recent writers, with the
exception of Jager ('99) in Taxus. I find the development of
cell-walls in the prothallium of Pinus to agree perfectly in its
early stages with that described by Sokolowa. Walls are
formed perpendicular to the wall lining the prothallial cavity,
thus each nucleus with its proper portion of the cytoplasm is
separated from all the other nuclei. No wall is laid down on
the inner sides of these cells, so that in radial section the cells
appear as uncovered boxes, the opening extending towards the
center of the prothallial cavity. In surface view the cells are
more or less isodiametric, polygonal in outline and very uniform
in size. A layer of densely reticulated cytoplasm surrounds
each nucleus, and delicate strands radiate from it to the ectal
layer of cytoplasm, thus giving a very different aspect to the
cytoplasm than it had prior to the development of cell walls
(figs. 160 and 161). Jager described the presence of walls on
the inner face of these cells in Taxus when the cells were first
organized, but other students have not confirmed his observa-
tions.
According to Sokolowa these cells grew inwards forming long
open tubes which extended to the center without division, a wall
was then formed at the inner end and the cells became divided
by cross walls. To these long cells the name alveoli was
applied. Only those from the sides extended clear to the center
before being closed, those from the extremities becoming more
or less wedge-shaped. Jaccard ('94) notes that inJSphedra some
of the alveoli may divide before reaching the center, but many
do not, while Arnoldi ('99 and '01) finds that no division occurs
in Sequoia until after the alveoli have met at the center and their
ends have become closed by walls. The development sub-
sequent to the formation of the open cells varies considerably
in Pinus from that described by these writers for other Gymno-
sperms. No cell has ever been observed to extend from the
86 MARGARET C. FERGUSON
circumference to the center of the prothallial cavity. The cells
are long, it is true, the walls delicate and wavy in outline, but
a ring of tissue composed of longer or shorter cells is formed
rather early in the inward growth of the prothallium. The cells
of the innermost row always remain open on their outer free
sides, their cytoplasm is more abundant than in the other cells
of the prothallium and their nuclei invariably retain a position
near the open side of the cells (fig. 162). As observed by
Jaccard ('94), and Jager ('99), the nuclei of the prothallium cease
to divide synchronously after individual cells have been organ-
ized. When the center is reached the cells close and thus, one
year after pollination, the endosperm becomes a solid mass of
tissue.
The prothallium grows rapidly after it has become a con-
tinuous cellular body and in a few days it fills all the central
and lower portion of the ovule. Above it is the prominent
nucellar cap, while only a few cells of the nucellus remain along
the sides separating the gametophyte from the integument (fig.
73, plate VII). Cell-divisions continue to take place, and the
cytoplasm becomes more abundant, though the prothallial cells
are never richly supplied with cytoplasm. Strasburger ('80),
Jager ('99), and several more recent students have noted many
nuclei in the endosperm cells. I have not observed multi-
nucleated cells in the prothallium of Pinus up to the time when
the suspensor has elongated and carried a several celled embryo
to a considerable depth into the endosperm. Later stages than
this have not been studied. There is often an appearance of
more than one nucleus in a cell, but careful study never fails to
demonstrate a delicate cell-wall between the nuclei. At an
early stage in prothallial development the cell-walls are very
delicate, scarcely more than condensations of the ectoplasm, so
that they might easily be mistaken, in Pinus, for strands of
cytoplasm. Doubtless the cells become plurinucleated during
a more advaned stage in embryo formation.
THE SO-CALLED SPONGY TISSUE.
The First Period of Growth. — When the macrospore-
mother-cell first becomes apparent it is surrounded by a group
LIFE HISTORY OF PINUS 87
of cells, three to five cells in thickness, which are more or less
clearly delimited from the surrounding tissue by their slightly
larger nuclei, their somewhat radial arrangement about the
macrospore-mother-cell as a center, and, in some instances, by
a rather indefinite and broken space which separates this group
of centrally lying cells from the adjacent nucellar tissue (fig.
124, plate XII). At the close of the tetrad-division these cells
have become much more conspicuous by the increase in the size
of their nuclei, the somewhat greater density of their cytoplasm,
and by the presence just exterior to them of an interrupted layer
of tabular cells which are evidently undergoing disintegration.
The disintegrating cells usually appear on one side first then at
other points about equally distant from the young gametophyte
(figs. 66, 69, plate VI ; 124, plate XII, 148, and plate XIV). It
was to this tissue, immediately surrounding the young endo-
sperm, together with the disintegrating cells just exterior to
it, that Strasburger gave the name " spongy " tissue, and for
convenience I shall use this term in speaking of it.
Ovules are frequently found during the summer and fall
which, so far as external appearances go, are perfectly normal,
but, when prepared for study, reveal the fact that either the
macrospore-mother-cell has never divided or the macrospore, if
formed, has not developed. Such ovules do not renew their
growth in the following spring. In those cases in which the
development of the mother-cell or of the young gametophyte
is arrested, very characteristic changes occur in the spongy
tissue. These cells grow and become rich in cytoplasm even
when the mother-cell does not divide, or when the macrospore
fails to germinate. But after a time they, too, become inactive,
their cytoplasm is gradually lost, their nuclei become dense
and deeply staining, and their cell-walls are very greatly thick-
ened (fig. 163, plate XV). This state of disintegration may
enter in at any time during the first period of growth but it is
more common before any divisions have occurred in the macro-
spore. When the mother-cell fails to divide, the cells of the
spongy tissue may grow until they almost equal it in size before
showing signs of breaking down. In such instances they bear
a very striking resemblance to the mother-cell, and might easily
88 MARGARET C. FERGUSON
be taken by one not familiar with the history of this tissue for a
group of macrospore-mother-cells (fig. 168, plate XVI). In
fig. 148, plate XIV, the slightly reduced cytoplasm of the cells
of the spongy tissue and the prominence of their cell-walls are
sure evidences that pathological conditions have entered in,
though all other parts of the ovule are still perfectly normal
the process of disintegration having only just begun. Had
this ovule been left in connection with the sporophyte for a
longer time, the spongy tissue would undoubtedly have assumed
later the character shown in fig. 163.
It is this abnormal appearance which I believe led Hofmeister
to conclude that there were two prothallia formed in the pines,
one for each season of growth. Strasburger thought that Hof-
meister mistook the normal spongy tissue for endosperm, and
Coulter and Chamberlain have recently expressed the same
view. Now the walls of the normal spongy tissue are never
thickened but remain even less prominent than those of the
nucellus. Hofmeister was surely too accurate a student of
cells as cells to have fallen into such an error. It is a well-
known fact that many ovules are organized in Pinus that never
reach maturity and they are very frequently found in the autumn
and late winter in the condition just described ; but with the
renewed growth of the healthy ovules in the spring, these fail
to develop farther and are soon detected by their smaller size.
Shortly afterward they become brown and dead. Having found
this thick-walled abnormal condition in the autumn and winter,
and in the spring finding within the ovules then developing the
large central cavity, it is not surprising that Hofmeister should
have concluded that a thick-walled transitory endosperm was
formed in the fall.
The Second Period of Growth. — When growth is renewed
in the spring the cells of the spongy tissue become organized
for the first time into a definite zone from two to three cells thick
which forms a hollow prolate spheroid immediately surrounding
the endosperm, and limited on its outer surface by a thin stratum
of disintegrating nucellar tissue. The cells and their nuclei are
not only somewhat larger than those of the nucellus, but their
most distinguishing characteristic is to be found in the greater
LIFE HISTORY OF PINUS 89
density of their cytoplasm which is almost identical with that of
the prothallium, while the cells of the nucellus are scantily
supplied with cytoplasm. These cells divide karyokinetically,
and, as they increase in number, they press against the adjacent
cells of the nucellus which become flattened against this con-
stantly advancing tissue, and are absorbed, only to give place
to other cells which meet a similar fate. Sometimes absorption
seems to precede the outward march of the spongy tissue, so
that this tissue is separated from the normal nucellus by a clear
space made up of cells of the nucellus which have lost all their
protoplasmic content, but which have not as yet suffered collapse
(figs. 157, 158, plate XV). The parietal layer of cytoplasm
which constitutes the endosperm remains always in closest con-
tact with the inner surface of this tissue (fig. 71, plate VI).
The cells of the spongy tissue are still prominent when the
endosperm becomes a solid multicellular body. Soon after-
wards, however, they show signs of disintegration, and at the
time of fertilization they have, as a rule, entirely disappeared
as cells, only the remnants of the cell-walls remaining. The
spongy tissue is then represented by a deeply staining fibrous
body of no definite structure which persists between the
gametophyte and the nucellus (figs. 162, plate XV, and 72,
plate VI; 73, plate VII).
The Nature and* Function of the Spongy Tissue. — The
prominent character of the cells surrounding the prothallium in
certain Gymnosperms has been commented upon, in a general
way, by all students of the Abietinece; but, as was noted by the
writer in 1900 and 1901 and confirmed by Coker in Taxodium,
1902, the true nature and function of these cells seem to have
escaped entirely the notice of previous writers, as they have in-
variably been described as tissue showing evidence of breaking
down. After a preliminary note regarding the nature of this
tissue was sent to press in 1900, Lang ('oo) described a similar
layer of cells about the endosperm in Stangeria. He designated
them as sporogenous cells and " possibly tapetal in nature."
As recently stated (1903), l these cells may possibly represent
sporogenous tissue, each cell being a potential macrospore-
1 See note at end of Appendix.
Proc. Wash. Acad. Sci., August, 1901.
9O MARGARET C. FERGUSON
mother-cell, but there is no evidence from the standpoint of origin
that such is the case in Pinus. They arise directly from a nucel-
lus in which a few days before their appearance every cell was
apparently like every other cell. This alone is not conclusive,
as the functional macrospore-mother-cell has a similar origin, so
far as one can see. But, what is more conclusive, the divisions
in this tissue are according to the typic method and present the
number of chromosomes characteristic of the sporophyte (figs.
164-167). If these cells were once, in some remote ancestor,
sporogenous in nature, they have entirely lost their primitive
function and have acquired a new and important function in
connection with the development of the endosperm. This is
not then a layer of disintegrating tissue, as described by all
earlier students of the Abietinece, but rather as already noted
by the writer (ipoi2) a definite zone of physiological tissue
which is intimately connected with the nutrition of the young
gametophyte. It doubtless not only passes on to the endosperm
the nutrititive substances derived from the nucellus, but is itself
active in the manufacture of food, as numerous starch grains
are often found within its cells. It is probable, too, that it
performs an important mechanical role in the way of protection.
It not only forms a support for the prothallium in its multinu-
cleated state, but gradually receding, it pushes before it, as it
were, the tissue of the nucellus thus making room within for
the growth of the delicate gametophyte.
Though we now know that this is a far more important tissue
than it was formerly thought to be, it does not seem to me wise to
apply to it the name tapetum or to suggest a new name by which
to designate it. Strasburger's term " spongy" tissue, although
given when the nature of this tissue was not understood and
being a misnomer so far as its structure and function are con-
cerned, has obtained a wide usage in the literature of the Gym-
nosperms, and should be retained, just as the term cell is still
retained in all biological literature.
DEVELOPMENT OF THE ARCHEGONIUM.
The Early Growth of the Arckegomum. — The archegonia
first become apparent during the latter part of May or the very first
LIFE HISTORY OF PINUS 9!
of June, the time varying somewhat with the species and with
the season. The degree of development which the prothallium
has attained when the archegonia-initials make their appear-
ance also varies not only in the different species but in the same
species. The differentiation of the archegonia may be deferred
until the prothallial cells have united to form a continuous tis-
sue ; but it quite as frequently happens that, while there still
remains a comparatively large, open space at the center of the
prothallial cavity, certain cells at the micropylar end of the pro-
thallium divide by periclinal walls more rapidly than do the other
cells of the endosperm and become comparatively rich in cyto-
plasm ; several of the superficial cells in this region do not so
divide, but continue to grow, and are distinguished from the
adjacent cells by their greater size, larger nuclei and more
vacuolate cytoplasm. These are the initial cells of the arche-
gonia (fig. 162, plate XV, and 169-171, plate XVI).
In less than a week after an archegonium-rudiment has ap-
peared, and while it is still quite inconspicuous, it divides, giving
rise to a small upper cell, the mother-cell of the neck, and a
large, lower cell which forms the venter of the archegonium
(figs. 171, 172, plate XVI). The small cell immediately divides
by an anticlinal wall, and the two cells thus formed divide by
walls that are perpendicular to the first, the resulting four cells
all lying in the same plane. These constitute what may be called
the normal neck in Pinus Strobus (figs. 173, 177, 180). Con-
siderable irregularity in the number and arrangement of the
neck-cells has, however, been noted even within the same spe-
cies. Frequently two of the four cells divide again, as figured
by Strasburger for Pinus Strobus in 1869, the six cells being
arranged in a single layer (figs. 178, 183, plate XVI, and 212,
plate XIX). Occasionally all four cells divide by anticlinal
walls, the neck then consisting of eight cells, all of which lie in
the same plane (figs. 179, plate XVI, and 213, plate XIX). In
rare instances the four cells divide by periclinal walls, when the
eight cells which compose the neck of the archegonium are dis-
posed in two tiers of four cells each (fig. 187, plate XVII).
This last represents the structure of the neck in Pinus sylvcstris
as figured by Mottier ('92) and Blackman ('98), and it is evi-
92 MARGARET C. FERGUSON
dently the usual condition in P. austriaca, P. rigida and P.
resinosa, but in these species, too, much variation obtains.
Variation in the number of neck-cells seems to be of common
occurrence in the Gymnosperms. It was first noticed by Hof-
meister in 1851 and has recently been discussed by Coulter and
Chamberlain ('01). Murrill (?oo) has figured considerable
irregularity in the number and arrangement of these cells in
Tsuga, while Coker ('02) shows a very marked variation in
Podocarpus.
At first the growth of the central cell is not followed by a
corresponding increase in the amount of protoplasm, so that its
cytoplasm early presents a very vacuolate appearance. There
may be one large, irregular central vacuole, or delicate strands of
cytoplasm may extend out from the nucleus to the ectoplasm,
these strands meeting and fusing at irregular intervals to form
vacuoles of various sizes. Thus a very beautiful pseudo-alveolar
structure is presented. Webber ('01) describes the cytoplasm in
the central cell in Zamia as representing at this time a foam struc-
ture of great beauty. I have never observed in this or any cell
in Pinus a cytoplasmic structure which, according to my inter-
pretation, could be designated as a true alveolar or foam struc-
ture in the sense in which Butschli ('94) uses the term. As the
central cell continues to enlarge its cytoplasm begins to develop
more rapidly, many strands extending out into and across the
vacuoles. Thus the size of the vacuoles is decreased while
their number is greatly increased. The central vacuole, if
present, may persist for a considerable time, or it may be re-
placed at once by smaller vacuoles (figs. 172-175). Gradually
the cytoplasm becomes more dense, and the vacuoles, receding
from the periphery of the cell, especially from its base and sides,
disappear last from its upper portion (figs. 176, 177). When
the ventral canal-cell is cut off, the vacuoles have nearly or quite
been replaced by a finely granular cytoplasmic reticulum in
which a greater or less number of larger, more deeply staining
granules are imbedded. These granules are frequently sur-
rounded by a clear court into which the protoplasmic network
has not extended. The number of the so-called proteid vacu-
oles is usually small at this time (fig. 178).
LIFE HISTORY OF PINUS
93
The nucleus of the central cell attains full size very soon
after its formation. It has a delicate, more or less interrupted
reticulum, and is characterized by a large vacuolate nucleus
which invariably occupies a central position. One or two
smaller nucleoli may also be present. This nucleus always
remains close beneath the neck-cells, as is the case in other
Gymnosperms, and, as a rule, is more or less concave on the
side toward these cells (figs. 172-177,181-183). As Blackman
has pointed out, the vacuolate nature of the cytoplasm renders
this nucleus very liable to displacement during the early stages
in the development of the archegonia, yet with well fixed ma-
terial it is always found in its normal position. Hirase ('95)
states that certain granules, which appear in the cytoplasm just
beneath the nucleus of the central cell in Ginkgo, have been
derived from this nucleus or from its nucleolus. Ikeno ('98),
also, describes the nucleus of this cell in Cycus as giving out a
granular substance during its growth period. No comparable
phenomenon has been observed in connection with the nucleus
of this cell in the species of pines which I have studied, but, as
above stated, the nucleus quickly reaches its mature size and
remains apparently unchanged until the inception of its division.
Very early in the history of the archegonium, the cells imme-
diately surrounding it become differentiated from the adjacent
endosperm-cells by their more regular form, the greater density
of their cytoplasm, and the increase in the size of their nuclei.
Thus a distinct sheath is formed about the venter of the arche-
gonium. This sheath usually consists of a single layer of cells.
It is more conspicuous in Plnus resinosa than in the other species,
and may become two cells broad at certain points, but even here
it is never two layered to any considerable extent. The nuclei
of these cells divide as the archegonium increases in size, the
axes of the spindles being always parallel with that face of the
cell which is adjacent to the egg. All the sheath-cells of a
given archegonium have several times been observed in the
same stage of mitosis, but this is very exceptional as these cells
do not ordinarily divide simultaneously. The sheath-cells
persist until after fertilization when they gradually lose their
cytoplasm and resemble the other cells of the prothallium.
94 MARGARET C. FERGUSON
Where adjacent archegonia crowd against each other these cells
early become distorted and partially destroyed. It is often diffi-
cult to demonstrate the presence of cross walls in the arche-
gonium-sheath. Neither have I been able to satisfactorily
demonstrate the presence of pores in the wall separating the
sheath-cells from the egg. Hofmeister ('6i-'62), Goroschankin
('80, '81), Arnoldi (bo), and Coulter and Chamberlain ('01) all
describe this wall in Pinus as thick and furnished with pores ;
but if such is the case it is not apparent in my material. On the
contrary the wall seems very thin and is scarcely differentiated
from the ectoplasm. It may be that further search on my part
will reveal both the " pits " and the " thickened wall," but thus
far I have not detected either.
No special attempt has been made to count the number of
chromosomes in the nuclei of the various parts of the sporo-
phyte and gametophyte, but whenever a nucleus was observed
in which the chromosomes were particularly clear and distinct
their number was always noted. In such cases twelve chromo-
somes have invariably been counted in the nuclei of the sheath-
cells. Chamberlain ('99) has found the same number in the
corresponding cells of Pinus Laricio. The early development
of the archegonium, as just described, agrees in the main with
that given by Strasburger in 1878.
As the archegonia grow the prothallium also continues to
increase in size, several layers of cells being formed above the
archegonia, except over their neck-cells. Here no prothallial
tissue is laid down, so that there arises an opening in the endo-
sperm leading from the neck-cells of each archegonium to the
nucellar cap (figs. 177-180). The presence of funnel-shaped
openings leading from the nucellus to the archegonia-necks in
Pinus was noted by Hofmeister in 1851 and their origin was
correctly described by him in 1862. In the last stages of pro-
thallial development preceding fertilization, the sides of this
tubular cavity often become very closely crowded together so
that the passage is obscured.
The number of archegonia in a single ovule varies in Pinus
Strobus, P. rigida and P. resinosa from one to five, the usual
number being three. In Pinus austriaca and P. montana var.
LIFE HISTORY OF PINUS 95
uncinata the number is larger, averaging about five. As
many as nine have been observed in a given prothallium in
Pinus montana var. uncinata. The form of the mature egg
depends largely upon the number and arrangement of the
archegonia. When there are not more than two or three, as is
frequently the case in Pinus Strobus, they may become almost
spherical in outline.
Division of the Central Cell. — As the central cell prepares
for division the cytoplasm between its nucleus and the neck-
cells is apparently resolved into fine granules, and there is a
more or less pronounced condensation of the cytoplasm about
the lower side of the nucleus. At the same time the nucleolus
disappears wholly or in part, the nuclear reticulum becomes
more open and broken, and the chromatin collects or condenses
at various places on the network (fig. 182). Soon a clear
court, similar to that described by Hof ('98), Fulmer ('98),
Nemec ('98 and '99), Strasburger ('oo) and others, makes its
appearance along the lower half of the nucleus. Inasmuch as
this nucleus is pressed close against the neck-cells such a court
does not arise along its upper side (figs. 183, 184). Delicate,
granular threads cross this court and press against the nuclear
membrane, while at the same time the upper and lower surfaces
of the nucleus become irregularly indented (fig. 185, plate XVII).
As the chromatin condenses to form the spireme, an achromatic
network, as already described for the corresponding stage in the
division of the generative nucleus in Pinus^ becomes apparent
in the nuclear cavity (figs. 182-185). When the spireme is
fully established it presents a beautiful nioniliform appearance,
and the longitudinal splitting of the band becomes apparent at
some points. The threads which arose earlier in the cytoplasm
seem at this time to have been again resolved into granules (fig.
186). Whether any of them enter the nuclear cavity and con-
tribute to the formation of the achromatic spindle has not been
definitely ascertained. The spindle, when formed, lies wholly
within the area previously occupied by the nucleus. Webber
(Joi) finds the origin of the spindle in the division of the gener-
ative cell in Zamia to be intranuclear. Farmer and Williams
('96 and '98) ascribe such an origin to the spindles studied in
96 'MARGARET C. FERGUSON
the Fucacea, and spindles of intranuclear origin have been
described by others. But while the achromatic figure in the
division of the central cell in Pinus comes to lie completely
within the nucleus, I would not claim that it is wholly of nuclear
origin ; if such were its source, the cytoplasmic activity in con-
nection with this division would be inexplicable. The earliest
stages in spindle-formation in this mitosis have not been ob-
served as yet, but when the transitional steps between the phases
represented in figs. 186 and 187 have been observed we shall
doubtless find that the cytoplasm has had some part to play in
the institution of the spindle. During the early metaphase of
the division the nuclear membrane can still be distinguished,
and clearly consists of a weft of threads (figs. 187, 188). I
have not observed any phenomenon in the prophase of this
mitosis at all comparable with the beautiful figure shown by
Murrill ('oo), as illustrative of the prophase of the division of
the central cell in Tsuga.
When the spindle arises, it is " multipolar in an axial plane "
and thus corresponds, with slight variation, to the mitotic figure
described by Duggar ('oo) in the microspore of Symflo carpus
fcetiduS) and by Wiegand ('99) in the microspore of Potamogeton
foliosus. In jPmus, however, the upper extremities of the
threads do not at first unite into groups, but remain practically
free, and are closely pressed against the neck-cells (fig. 187).
The several poles, formed at the inner or lower extremity of the
karyokinetic figure, soon draw together forming a single, very
sharply defined pole ; or the fully developed spindle may remain
more or less truncate at its lower end. Blackman describes
this spindle as bluntly truncate at both extremities. I have fre-
quently observed such a spindle during a late anaphase of the
division, but this is only one of the various aspects which may
be presented during metakinesis and later stages in this mitosis.
The upper extremities of the achromatic spindle-fibers may
never draw together at all ; they may unite to form two or more
poles ; or they may give rise to one pole which may be blunt
or very slender (figs. 190-194). But whatever form may be
assumed by this spindle during the later stages in its develop-
ment, there is always formed, at an early period, a diarch spindle
LIFE HISTORY OF PINUS 97
which is multipolar at one extremity and monopolar, or nearly
so, at the other (figs. 187, 188). A similar figure is also organ-
ized in the mitoses which occur in the development of the pol-
len-grain, and at an early stage in the division of the generative
nucleus in the pines, as already described in this paper ; and it
is suggested that such a figure may be characteristic, at least in
the higher plants, of those indirect divisions which result in the
formation of nuclei or cells of unequal size.
The chromosomes, when oriented at the nuclear plate, are in-
variably in the form of U's or Vs. Blackman states that they
are straight rods but he does not so figure them. The cell-
plate, during the early stages in its formation, lies midway be-
tween the developing nuclei, but when the daughter-nuclei are
fully formed, the nucleus of the oosphere is, as a rule, farther
removed from the cell-plate than is the nucleus of the ventral
canal-cell. A prominent cell-plate is formed and the plane of
cleavage separating the ventral canal-cell from the egg becomes
evident in many instances before the disappearance of the
spindle. As Chamberlain ('99) has shown, the lower portion of
the spindle at this time is ordinarily convex, while the part
within the ventral canal-cell is concave (figs. 195-197, plate
XVII, and 200, 201, plate XVIII).
I was able in several preparations similar to that illustrated
in fig. 191 to count the number of chromosomes, and twelve or
thirteen were found in both groups instead of eight as counted
by Dixon ('94).
The Ventral Canal-cell. — According to my observations, a
definite wall, separating the canal-cell from the egg-cell, is
always formed in Pinus. Coker has made the interesting
observation that no wall is developed in Podocarpus, the nucleus
of the ventral canal-cell lying free in the egg.1 As a rule the
nucleus of the ventral canal- cell in Pinus does not present a
normal appearance, but shows signs of disintegration very early
in its history. It is doubtful, in some cases, if a nuclear mem-
brane is ever formed, and there are probably instances in which
fusion of the chromosomes never takes place at all. The kar-
yokinetic structure shown in fig. 193 would very presumably
, : J See note at close of Appendix.
98 MARGARET C. FERGUSON
give rise to such a nucleus, if we may so denominate it, as that
illustrated in the ventral canal-cell of fig. 196 ; although Black-
man, judging from such a figure as that portrayed in fig. 194,
considers it impossible that the chromosomes of the ventral
canal-cell should ever fail to fuse. The nuclear membrane,
when present, very soon breaks down, and the chromatic sub-
stance becomes scattered throughout the cell (figs. 198-202).
This cell immediately preceding and at the time of fertiliza-
tion ordinarily forms a deeply staining mass which lies just
beneath the neck-cells and above, but in contact with, the egg
(figs. 180, plate XVI, 202, plate XVIII, and 213, 215, plate XIX).
Rare exceptions to the rapid disintegration of the canal-cell have
been observed and will be described in the appendix to this
paper. But in the study of several thousand archegonia of
Pinus Strobus no instance has been found in which the nucleus
of the egg and of the ventral canal- cell were similar in form.
The nearest approach to a normal nucleus that has been observed
in the ventral canal-cell of this species is that shown in fig. 197,
plate XVII. Occasionally this cell is somewhat enlarged and is
furnished with a rather scanty amount of cytoplasm in which
distinct chromosomes, or chromatic figures of various forms are
imbedded. Of the many variations that have been found to
occur in the structure of the ventral canal-cell in the mature
archegonium but two have been illustrated — figs. 199 and 199,
plate XVIII. It is probable that in such instances a true nucleus
has ever been formed if, indeed, the chromosomes have fused
at all. The character of the cell at this time is such as to pre-
clude the possibility that a division of this cell is being initiated.
There seems to be a definite relation between the structure of
the ventral canal-cell and the character of the upper part of the
mitotic figure formed in the division of the central cell. This
is plainly demonstrated by a comparison of figs. 190 to 197,
plate XVII, and 200-202, plate XVIII. Figs. 190, 193, 196
and 202 represent an especially interesting series.
The separation of the canal-cell from the cytoplasm of the
oosphere, as Strasburger ('72) and Blackman ('98) have de-
scribed in Pinus, is, I believe, due to a shrinkage of the egg-
cytoplasm caused by imperfect fixation ; and it is possible that
a similar appearance in Cycas, Ikeno ('98), has a like origin.
LIFE HISTORY OF PINUS 99
MATURATION OF THE EGG.
The Descent and Growth of the Egg-nucleus. — The egg-
nucleus is no sooner formed than it begins to increase in size,
becoming greatly enlarged even before the disappearance of the
spindle-fibers (figs. 196-202). As the nucleus moves toward
the center of the oosphere, threads of more or less delicacy
extend, in a radial manner, from its wall into the surrounding
cytoplasm. These fibers are not equally well defined in all
preparations, but, whatever the degree of their prominence,
they are invarably more strongly differentiated about the upper
side of the nucleus, and may extend from the nucleus to the
top of the egg (figs. 202-204).
As already stated, few, if any, vacuoles persist within the
the venter of the archegonium at the time of the division of the
central cell. Following their disappearance, there arise numer-
ous spherical bodies, the so-called proteid vacuoles. Coordi-
nate with the downward movement of the egg-nucleus, these
bodies assume a position about the periphery of the oosphere,
more especially at its base (the organic apex of Strasburger),
and at its sides (figs. 179, 180, plate XVI, 214, plate XIX). Un-
der a low power, the cytoplasm of the mature egg appears dense
and finely granular; the " proteid vacuoles" do not seem to
differ materially from the protoplasm in which they are im-
bedded ; and many deeply staining granules are scattered
throughout the cell. With greater magnification, however, a
very beautiful, granular reticulum becomes apparent. There
is no suggestion of the alveolar structure described by Biitschli
('94). At times this reticulum is everywhere crossed by short
fibers which have no definite arrangment and are, apparently,
not confined to any fixed period in the history of this cell (fig.
200). The spheres in the outer and basal portions of the cyto-
plasm are resolved into very complex structures which, although
they simulate the appearance of nuclei, could never be mistaken
for such bodies by one familiar with cell-structures (figs. 202,
203.)
No cytoplasmic radiations, similar to those described by
Belajeff ('91) in Taxus baccata, and by Dixon ('94) in Pinus
sylvestriS) have been observed in connection with the fully
IOO MARGARET C. FERGUSON
developed egg-nucleus in any of the species of pines which I
have studied.
During the growth and downward movement of the egg-
nucleus, it never presents, in Pinus Strobus, a definite network,
such as is observed in the nucleus of the ordinary resting cell ;
but it is characterized at a very early date by an open, inter-
rupted reticulum, on which are arranged irregular granules of
various sizes. This meshwork may be extremely delicate ; it
may assume a heavy appearance ; or it may become very much
interrupted and broken, many detached portions lying loose within
the nuclear cavity (figs. 196, plate XVII, to 205, plate XVIII).
The egg-nucleus of Pinus austriaca and P. montana var. unci-
nata, may frequently show from an early date a beautifully regu-
lar reticulum (fig. 269, plate XXIV). Nucleoli have rarely been
observed in this nucleus in Pinus Strobus during the first stages of
its development (figs. 196, 199 and 200-201) ; but in Pinus aus-
triaca they occasionally arise very early (fig. 195). When the
nucleus has attained considerable size, small, nucleolus-like
bodies, containing a single central vacuole, appear in connection
with the nuclear net ; and at the same time a slightly larger nu-
cleolus is observed in the lower part of the nucleus, usually in
connection with its membrane (fig. 202). As the nucleus con-
tinues to grow, this nucleolus also increases in size, gradually
becoming large and very vacuolate (figs. 203-205).
When the egg-nucleus reaches maturity, it has attained huge
dimensions, and its outline, depending on the form of the egg,
is spherical or elliptical. The nucleolus, if demonstrable, is
always found in the lower part of the nucleus ; and there are
usually several smaller bodies, designated in this paper as sec-
ondary nucleoli, scattered throughout the nucleus (fig. 205).
These secondary nucleoli are invariably found in connection
with the reticulum, but, as Montgomery ('98) believed regarding
apparently similar structures, they are probably caught in, not
vitally united to it. They may be present in great abundance,
or they may be entirely absent from the nucleus. The reticu-
lum, on which the chromatic substance is disposed, presents
numerous aspects, as already indicated in the description of this
nucleus during its period of growth. Under very high magni-
LIFE HISTORY OF PINUS IOI
fication, it does not show, in normal conditions, a true granular
structure; but it may present a most delicate, interrupted,
granular network ; or, it may consist of large, irregular, dif-
fusely-staining masses which are united into an imperfect reticu-
lum (figs. 206, #, and 206,^). In the latter instance the
chromatic granules are either too minute to be distinguished, or
they have been dissolved in the linin ground-work. The linin,
always very abundant in this nucleus, may form heavy hyaline
cords, on which the chromatin is collected at irregular intervals
(figs. 206, £, and 206, f) ; but it more often consists of less con-
spicuous strands (figs. 206, 3, to 206, d). Great as are the vari-
ations in the structure of this nucleus, its chromatin has always
been found, in the species of pines which I have studied, to
exist either in the form of irregular granules of varying sizes,
or apparently dissolved in the liniri. Such a resolving of the
chromatin into nucleoli as that described by Chamberlain ('99)
in Pinus Laricio and illustrated in his figs. 14 and 15 has not
been observed in normal nuclei by the writer.
Whether the various appearances presented by the egg-nucleus
represent normal phases in its life history, or whether one is
normal and the others are artifacts resulting from the action of
fixing agents, is, of course, a mere matter of conjecture. But,
inasmuch as these different aspects are characteristic of this
nucleus during its period of growth, also after it has to all
appearances reached maturity, and again at the time of its con-
jugation with the sperm-nucleus, it seems reasonable to conclude
that all are normal and correspond to definite physiological
processes, which take place within the nucleus. Hertwig's
('98) interesting experiments on fed and unfed Actinosphcerium
are in point here. They seem to show conclusively that the
structure of a nucleus varies with the character of the work
which is being done by it.
Strasburger ('84) described the nucleus of the oosphere in the
Abietmece as being densely filled with a granular substance which
entirely obscured or masked the chromatin. This substance he
called metaplasm, and virtually considered the nucleus a vacuole
filled with a nuclear sap capable of taking up or elaborating this
material. Ikeno found a similar substance in the sexual nuclei
IO2 MARGARET C. FERGUSON
in Cycas in 1898 and more recently in Ginkgo ('01), and Arnold i
('oo) in Cephalotaxus. Blackman ('99) devoted several para-
graphs to a discussion of metaplasm, as it manifested itself in
the egg-nucleus of Pinus sylvestris. He found that it was
present in the young nucleus in the form of granules, but that
it later united with the chromatin to form the nuclear reticulum.
Chamberlain ('99) does not recognize the presence of this sub-
stance in the egg-nucleus in Pinus Laricio ; and there is no
evidence of its existence in the sexual nuclei of the species of
pines which I have studied.
According to Wilson ('99) "protoplasmic substances repre-
sent the active, metaplasmic structures the passive elements " of
the cell. During the development of the egg-nucleus in the
species of pines which have formed the basis of these studies,
there is never any deposit within the normal nucleus of a granu-
lar substance; but the linin, as already stated, becomes very
abundant. Just what proportion of it is active in cell division,
we are unable to say. Without doubt a large part of the linin
merges into the cytoplasmic network during the first segmen-
tation of the oosphere-nucleus, but even so, it can not be classi-
fied with the passive elements of the cell.
Blackman ('98) wrote: "The stage in which the nucleus is
found in a position between the apex and the center of the egg
is rarely met with" ; and Chamberlain ('99) stated " that in over
three hundred preparations, less than a dozen " show early
stages in the development of the egg-nucleus. During the
course of these investigations upon the pines, about four thousand
preparations, representing many thousand archegonia, have been
studied, and no developmental stage has been more frequently
met with than that by which the nucleus assumes its central posi-
tion in the egg. Such an appearance as that illustrated by
Chamberlain in his figs. 18 and 19 has been observed in both
the young and the mature egg-nucleus, in the conjugating nuclei,
and also in the various nuclei of the proembryo. They have
been wholly disregarded in the present discussion of the matura-
tion of the egg, for, in my material, these figures, and also
Blackman's figure n, would be interpreted as representing dis-
integration stages. Every step has been repeatedly traced from
LIFE HISTORY OF PINUS IO3
the ordinary nuclear reticulum, to nuclei which can scarcely be
distinguished from the surrounding cytoplasm, and then to arche-
gonia, which appear perfectly normal except that no nuclei can
be demonstrated within them. It is a well known fact, already
commented upon in this paper, that the number of seeds derived
from a pine cone is very small in comparison with the number
of ovules formed in the same cone. An examination of fresh
material shows that development may cease at any point be-
tween the early stages in the formation of the ovule and the last
steps in the ripening of the seed. This cessation of growth
effecting first individual cells does not at once become apparent,
and so cannot be avoided, in its earliest stages, when one is
putting up material for cytological work. Under such condi-
tions, it is inevitable that, with a limited amount of material, the
abnormal will be interpreted for the normal.
The entire development of the archegonium in Pinus is passed
through in about two weeks, probably not more than five days
elapsing between the cutting off of the ventral canal-cell and
fertilization. In Pinus montana var. uncinata these processes
are apparently much more closely united in point of time, as
the pollen-tube, in some cases, has reached the endosperm
before the division of the central cell is complete (fig. 207,
plate XIX).
The Proteid Vacuoles. — The true nature of the proteid
vacuoles is a subject which attracted my attention very early in
the course of these investigations. There can be no doubt that
there is an intimate relation between the sheath-cells of the
archegonia in the pines and the substance of the egg, such as
is believed to exist between the follicle-cells and the egg in ani-
mals. But the exact nature of this connection in Pinus is not
easily determined. I have rarely examined a preparation show-
ing archegonia without studying the relation of the sheath-cells
to the oosphere ; and yet no entirely satisfactory evidence, be-
cause not demonstrable beyond a question, of the origin and
nature of the so-called proteid vacuoles has been found.
Hirase ('95) observed that the granules in the egg of Ginkgo
were of nucleolar origin, being derived both from the nucleus
of the central cell and from the nuclei of the sheath-cells.
IO4 MARGARET C. FERGUSON
Arnold! ('oo) found that substantially the same thing was true
in Cephalotaxus. He was not able to detect the passage of the
nucleoli from the sheath-cells into the egg, but, since these
granules were present on both sides of the membrane of the
egg-cell he accepted the fact of their transference. I have
frequently seen a nucleolus partly without and partly within the
nucleus of a sheath-cell ; but in no instance could I be sure that
such a condition was not the result of mechanical displacement.
Ikeno ('98) found direct evidence that the nutritive spheres in
Cycas are of nuclear origin. But no such phenomena as he
observed in Cycas occur in Pinus. Platner ('86) described the
passage of the follicle-cells into the ovum in Helix, and a few
other such instances have been recorded in animals. Arnoldi
('oo) has recently noted a most remarkable migration of whole
nuclei from the sheath-cells into the egg in several species of
pines. He has observed, in a single series, as many as one
hundred and fifty nuclei passing into the ovum. From the fact
that Arnoldi writes " Strobus" in a parenthesis after Pinus
Peuce, I infer that he employs the terms as synonyms ; but I
find no authority for such a usage, and cannot accept his con-
clusions as holding good for -Pinus Strobus. It does not seem
possible that, in a careful examination of several thousand
archegonia, so obvious a phenomenon as that described by
Arnoldi could have % escaped detection; and I must, therefore,
conclude that it does not take place in the species of pines
which I have studied. I fully believe that the sheath-cells play
an important role in the nutrition of the egg ; but it is the
method by which this is accomplished, as described by Arnoldi,
that I cannot accept for the species of pines studied. Coulter
and Chamberlain ('01) not only accept Arnoldi's observations
for Pinus but describe a like phenomenon in Cycas. Basing
their statement on the results of Ikeno's studies, they record, on
page 22, the following surprising fact with reference to Cycas:
"The contents of the jacket-cells, nuclei and all, now pass
through the pores into the central cell." I find no authority
for such a statement in Ikeno's paper. If I correctly translate
the German, Ikeno describes neither the transmission of the
nucleus nor of the cytoplasm from the sheath-cells into the egg,
LIFE HISTORY OF PINUS
but he does note a most interesting transfer of nuclear sub-
stance, that is, a substance secreted by the nuclei, from the
nuclei of the sheath-cells into the cytoplasm of the egg. In
the course of his discussion Ikeno says : " Bemerkenswerth ist
es ferner, dass der Zellkern der Wandungszelle haufig sich der
Centralzelle nahert und dort einen nach dem nachsten Plasma-
faden gerichteten kurzen Schnabel bildet (fig. 6). In einen
andern Fall beobachtete ich, das der Zellkern der Wandungs-
zelle sich bis an die Cellulosemembran begiebt, welche an die
Centralzelle angrerizt und mit dem ganzen Korper an diese sich
anlegt (fig. 7, #, b). Offenbar sollen alle diese Vorgange den
Uebergang des in diesen Zellkernen enthaltenen Stoffes nach
der Centralzelle erleichtern." So far as I am aware then,
Arnoldi is the only investigator who has observed the passage
of entire nuclei into the egg in the Gymnosperms.
Some interesting observations have been made during this
study regarding the nature of the nucleolus of the egg-nucleus.
As already indicated this nucleolus does not arise in Pinus
Strobus until the egg-nucleus has attained considerable size.
It appears in the lower part of the nucleus as a minute, solid,
spherical body ; during growth a small central vacuole appears,
then other vacuoles, until, at maturity, it is completely filled
with vacuoles of various sizes (figs. 202-205, plate XVIII).
A limiting membrane is not always apparent in this nucleolus
(fig. 208, plate XIX; but in some instances, there seems to be
very strong evidence of such a membrane (figs. 205 and 209).
In fig. 205 the nucleolar wall has been broken at one place
and a vacuole, lying near the point of rupture, has been in-
dented along its outer surface, thus becoming crescent shaped.
Montgomery ('98) sounded a word of warning against inter-
preting the peripheral stratum of the ground substance of the
nucleolus as a wall layer; and there is a possibility that, in the
figures above referred to, what appears like a limiting mem-
brane is only the outer unmodified portion of the nucleolus.
The attitude of this nucleolus toward dyes varies much at
different periods in its history. It may or may not take the
safranin stain characteristic of Flemming's triple combination ;
it may stain intensely with gentian-violet or iron haematoxylin
Proc. Wash. Acad. Sci., August, 1904.
IO6 MARGARET C. FERGUSON
(figs. 205 and 208) ; it may show a weak reaction to these stains
(fig. 209), or it may be absolutely unaffected by them, remaining
as a hyaline or greenish yellow structure (fig. 210). When the
nucleolus resists the action of dyes, its nucleus is usually totally
free of the secondary nucleoli, which have been described in
connection with the maturation of the egg-nucleus, and the
cytoplasm of the egg is studded, to an unusual degree, with
large, deeply staining granules. But the nucleus containing a
nucleolus which stains with avidity, generally contains, also,
innumerable secondary nucleoli ; at the same time, there are
comparatively few deeply staining granules in the cytoplasm
of the egg.
The position of the secondary nucleoli with reference to the
primary nucleolus is frequently such as to indicate that the
former originate in the latter (figs. 227, plate XX, and 208,
plate XIX). The only observations which would militate
against such an origin are the few cases found in which the
secondary nucleoli seem to appear earlier than the primary
nucleolus (fig. 195, plate XVII). It may be that, in these cases,
the primary nucleolus has not yet become differentiated in
structure from the secondary nucleoli, as would evidently be
true in a stage slightly younger than that shown in fig. 202,
plate XVIII ; or it may be true that the primary nucleolus is pres-
ent, but fails, at this time, to stain. Floderus ('96) describes a
somewhat similar origin of the paranuclei, in Tunicates, from
the nucleolus proper.
The nuclei of the cells surrounding the archegonia contain
from three to five nucleoli, and one or more nucleolus-like struc-
tures may be present in the cytoplasm of these cells. Each
nucleolus is surrounded by a clear court which, as Zimmermann
('96) has pointed out, is evidently not an artifact. Debski ('97)
opposes this view, however, and considers the clear court to be
attributable to the shrinkage of the nucleolus, since he does not
find it when material is treated with xylol instead of cedar oil.
These nucleoli may be spherical, elliptical, irregular, or long
and almost dumbbell-like in outline. The ordinary cells of the
prothallium do not show nucleoli. If such bodies be present in
these cells they are small and obscured by the nuclear reticulum.
LIFE HISTORY OF PINUS IO7
At about the time of the cutting off of the ventral canal-cell
many small nucleolus-like masses appear in the nuclei of the
sheath-cells — twenty or more occurring in a single nucleus.
When the egg has reached maturity, and during the later stages
of its history, no nucleolus, or but one or two nucleoli, can be
demonstrated in the nucleus of a sheath-cell. These nucleoli
are no longer surrounded by a hyaline court, but are imbedded
in the chromatic network.
The nucleoli of the sheath-cells present the same attitude
toward stains as does the nucleolus of the egg-nucleus. But
while the nucleoli of the sheath-shells frequently stain but feebly
they rarely fail entirely to stain.
Similar color reactions have been observed in connection with
the nucleoli, as already described, in the microspore-mother cell
of Pinus. The occurrence of unstained nucleoli in the same
nucleus in which others were deeply colored is common in the
microspore-mother-cells especially at about the time of synapsis.
I am aware that conclusions based upon staining reactions alone
are not to be trusted, but when accompanied, as here, with other
phenomena they may be highly significant.
The nucleolus of the egg-nucleus and also the nucleoli of
the sheath -cells in Pinus appear to represent active portions of
the cell rather than inert masses of matter. Certain aspects
presented by these nucleoli are surely suggestive of plastids.
The uncolored framework of the egg-nucleolus reminds one
very strongly of a chlorophyll body from which the pigment
has been extracted. Yet we would not, in the present state of
our knowledge, denominate them plastids. I believe, however,
although the phenomena are not of such a nature as to admit of
definite demonstration, that the nucleolus of the egg-nucleus,
and also the nucleoli of the sheath-cells are actively engaged in
the formation of a substance which in the egg-nucleus, at least,
assumes the shape of secondary nucleoli. These nucleoli be-
come diffused throughout the nucleus, from which they pass,
probably in solution, into the egg cytoplasm. Here they are
again differentiated, and by a gradual development, give rise
to the " proteid vacuoles " or nutritive spheres of the oosphere.
It may be that the greater size of the egg-nucleus, in com-
108 MARGARET C. FERGUSON
parison with that of the sperm-nucleus, is correlated with the
physiological role, as above suggested, which it plays in the
cell. We cannot, here, enter into a discussion of the volu-
minous literature dealing with the origin, function, and destiny
of the nucleoli ; but a few of the many views which have been
advanced may be noted.
Strasburger ('95, '97 and 'oo) expresses his conviction that
nucleolar substance contributes to the formation of spindle-
fibers. A similar view is held by Fairchild ('97), Harper ('97),
Debsky ('97), and other students of the Bonn Laboratory, and
by Nemec ('99), Farmer ('94) and others. Strasburger ('95)
also sees indications of a connection between the nucleolus and
the cell-plate and he has recently ('97 and 'oo) sought to show
that the nucleoli make active the spindle-forming substance in
the cytoplasm, or that they enhance the activity of the kino-
plasm.
Flemming ('82), Humphrey ('94), Zimmermann ('95), Sar-
gant ('96 and '97), Duggar ('99), Mottier ('oo), and many others
believe that the nucleoli represent reserve supplies of chromatin.
Dixon ('99) finds in them a vehicle of inheritance. Hirase ('98)
thinks that they give rise to the attractive spheres ; and accord-
ing to Karsten ('93), Lavdowsky ('94) and Wilcox ('95) they are
centrosomes. Rosen ('95) considers that the nucleoli are equal
in dignity to the chromatin, that they have no connection with
the centrosome and that they do not serve to nourish the chro-
mosomes.
Jordan ('93) states that " their function is almost certainly one
of nutrition either concerned in the storage or elaboration of
nutritive material " and believes that there is substantial reason
for looking upon the nucleolus wherever found as concerned .in
one way or another with the active metabolism of the cell.
Lukjanow ('88) and Macallum ('91) consider the nucleoli to be
excretory organs which are intimately related to the nutritive
spheres of the egg, these spheres arising through a process of
deposition from the nucleolus. And Hacker ('93) observes that
the nucleolus is a contractile vacuole which absorbs proteid
substances ; the absorbed materials undergo a chemical change
within the nucleolus and are then periodically discharged.
LIFE HISTORY OF PINUS
Flemming ('82), Zacharias ('85) and Zimmerman ('93) ascribe
to the nucleolus the dignity of a nuclear organ ; and Mont-
gomery ('98) makes the following suggestion: "That though
the nucleolus consists of substances which stand in some rela-
tion to the nutritive processes of the nucleus, and so, at the
time of its formation, may be a functionless inert mass of
matter, yet it may at later periods in the history of the resting
nucleus, acquire some active function, and thus gradually come
to acquire the value of a nuclear organ."1
Obst ('99) remarks that the significance of the nucleolus is
truly dark, but he considers it to be in some way the result of
chemical action whose cause must be sought in the physiolog-
ical processes of the cell. A glance at the theories regarding
the nature of the nucleolus as briefly outlined above is certainly
sufficient to confirm Obst's conviction that our knowledge of
the origin and function of the nucleolus is still very imperfect.
Yet it cannot be doubted that we have in the nucleolus not
merely a mechanical store-house, but a structure which is inti-
mately connected with the vital activities of the cell. We still
have in the nucleolus a most attractive field for investigation,
and the best cytological, physiological, and microchemical
technique must be brought to bear upon the problem before
we can hope to understand aright the true nature of this
structure.
The Receptive Vacuole. — Immediately preceeding fertiliza-
tion a cavity appears in the egg-cytoplasm, just beneath, or in
the near vicinity of, the neck-cells (figs. 211, 213, 214, plate
XIX). In some cases this opening may not arise until the instant
of fertilization. This cavity, which was thought by the earlier
writers to represent the lower portion of the pollen-tube within
the oosphere, has been explained by Blackman ('98) as due to
the sudden inrush of the contents of the pollen-tube, and by
Arnoldi ('oo) in Cephalotaxus, as caused by the downward
movement of the conjugation-nucleus. Shaw ('98) suggests
that the concavity in the upper part of the egg in Onoclea, just
prior to fertilization, may correspond to the receptive spot ; and
there is every evidence that in Pinus this opening in the cyto-
1 See note at close of Appendix.
IIO MARGARET C. FERGUSON
plasm represents the last act of the egg in its preparation for
the reception of the sperm-nucleus. If it were formed by the
movement of nuclei or other bodies through the protoplasm, we
should expect the cytoplasm to draw together again, as during
the downward movement of the egg-nucleus ; but, in reality,
this opening persists throughout the entire later history of the
archegonium. Following fertilization it is sometimes found at
one side or a little below the neck of the archegonium. This
position is doubtless due to displacement at the time of con-
jugation (fig. 215). The regular clear outline of this cavity,
together with the fact of its presence in the unfertilized as well
as in the fecundated egg, warrants one in considering it a
definite character of the mature oosphere.
I have suggested the name, receptive vacuole, for this vacu-
ole which is such a constant feature of the egg at the time of
fertilization and immediately prior to conjugation. The pollen-
tube suddenly empties into the archegonium a large amount of
material — several nuclei, a comparatively large amount of
cytoplasm (see pollen-tube, fig. 120, plate XII, and fig. 214,
plate XIX) and considerable starch. The sudden acquisition of
this matter by an already densely filled egg might from the
increased pressure alone, cause fatal results. That the egg
should thus prepare for the reception of the sperm-cell is not
only a very beautiful, but a very interesting illustration of the
economy so often observed in nature.
SUMMARY.
After a period of growth the macrospore germinates and by
a typic division gives rise to the first two nuclei of the female
gametophyte. These usually pass to opposite poles of the pro-
thallial cavity and soon divide again. Divisions follow rather
leisurely during the fall, all the nuclei dividing synchronously.
After thirty-two or more free nuclei are formed the long period
of rest is entered upon.
In early spring nuclear division is resumed and a large num-
ber of nuclei are formed ; about two thousand have been counted
at the time when cell-walls are first laid down.
Walls are first developed in the prothallium during the latter
LIFE HISTORY OF PINUS III
part of May. The nuclei are thus separated, but no wall is
formed over the inner surface of the prothallium so each nucleus
is, as it were, enclosed in an open box. These cells stretch
out toward the center but never reach it without having first
divided by cell-walls from two to several times. The innermost
layer of cells always remains open on its free side until the cells
meet in the center and the endosperm becomes a continuous
cellular body.
The spongy tissue becomes apparent as soon as the macro-
spore-mother-cell is differentiated, but it is not organized into a
definite zone with sharply defined limits until the beginning of
the second season of growth.
These cells function as a physiological tissue of great impor-
tance in the nutrition of the young gametophyte. They doubt-
less convey nutrition derived from the disintegrating adjacent
nucellar tissue to the endosperm and are also occupied in the
manufacture of food materials. The spongy tissue doubtless
further serves to protect the young prothallium, not only by
affording support but by driving out, as it were, the nucellar
tissue, thus making room for the delicate female gametophyte.
The time at which the archegonia appear varies somewhat,
but in general they can first be detected about two weeks before
fertilization. They are normally found at the micropylar end
of the prothallium, and arise by the differentiation of certain
of the peripheral cells. By the later growth of the female
gametophyte, the mature egg is sunk to a considerable depth
in the prothallial tissue, but there always remains an open
channel leading from the neck-cells to the nucellar cap. The
number of archegonia varies in the different species from one
to nine. When the number of oospheres formed is small they
are almost spherical in outline ; but this shape may be greatly
modified according to the number and arrangement of the
archegonia.
In Pinus Strobus the typical neck of the archegonium con-
sists of four cells, all lying in the same plane, while in Pinus
austriaca and P. rigida it is made up of eight, disposed in two
layers of four cells each ; but there is a lack of uniformity both
in the number and in the arrangement of these cells, not only
in different but in the same species.
112 MARGARET C. FERGUSON
The central cell is very vacuolate at first, its nucleus always
remains close beneath the neck-cells and is more or less con-
cave on the side toward those cells. When the ventral canal -
cell is cut off, about a week before fertilization, the vacuoles
have nearly disappeard from the venter of the archegonium.
The spindle in the division of the central cell arises as a
multipolar diarch figure and apparently lies wholly within the
nucleus. That portion of the mitotic figure which gives rise to
the ventral canal-cell varies much in the later stages of its de-
velopment ; but, whatever irregularity characterizes this part of
the spindle, it always becomes monopolar or nearly so, at its
lower, inner extremity.
The form and structure of the nucleus of the ventral canal-
cell are very variable, and are correlated with the irregularities
occurring in the upper, outer portion of the achromatic spindle
during the division of the central cell. There are probably in-
stances in which no membrane is developed about this nucleus :
in such cases the chromosomes never fuse to form a network.
The ventral canal-cell rarely presents the appearance of a nor-
mal cell ; at the time of fertilization it usually persists as a
small, somewhat crescent-shaped, deeply staining body which
lies just beneath the neck-cells of the archegonium and above,
but in contact with the cytoplasm of the egg.
As the egg-nucleus assumes its central position in the oosphere,
it increases much in size, and many fibers arise in the cytoplasm
surrounding it. These threads have, in general, a radial
arrangement and are more prominent along the upper side of
the nucleus. The structure presented by the growing, and also
by the mature, egg-nucleus may vary from a most delicate net-
work bearing minute granules to an interrupted, imperfect
reticulum composed of large, irregular, diffusely staining ele-
ments. These various aspects are doubtless the expressions of
the different physiological activities with which this nucleus is
concerned. The normal egg-nucleus has one large, vacuolate
nucleolus and a variable number of small, secondary nucleoli.
There is no evidence of the presence in this nucleus of a special
metaplasmic substance.
During the maturation of the egg, many nutritive spheres
LIFE HISTORY OF PINUS 1 13
arise in its cytoplasm. At first these are irregularly scattered
throughout the cell, though more prominent at its periphery ;
in the mature egg, they are largely confined to the peripheral
portions of the lower half of the cytoplasm. It is suggested,
though not definitely demonstrated, that these nutritive spheres
are the products of nucleolar activity, having originated within
the nucleolus of the egg and the nucleoli of the sheath-cells.
The egg-cytoplasm presents a delicate reticulum, in which, at
times, fibers occur. Immediately preceding fertilization, an
opening arises in this cytoplasm, just below or in the near
vicinity of the neck-cells. This cavity is apparently formed for
the reception of the sperm-cell, and the name " receptive
vacuole " has been applied to it by the writer.
CHAPTER IV.
FERTILIZATION AND RELATED PHENOMENA.
CONJUGATION.
The Coming Together of the Gametofhytes. — When the
time for fertilization arrives the pollen-tube has forced its way
between the neck-cells of the archegonium and stands just above
the egg (fig. 120, plate XII), but it does not under normal con-
ditions enter the archegonium. The fact that the pollen-tube in
Pinus does not penetrate the egg has recently been observed by
Blackman ('98), and Coulter and Chamberlain ('01). Standing
just above the egg, the apex of the tube is ruptured and almost
all of its contents passes into the cytoplasm of the egg. The
sperm-cell with its dense cytoplasm and two nuclei, the tube-
nucleus, the stalk-cell, a part of the cytoplasm from the pollen-
tube, and some of the starch grains from the male gametophyte
can all be distinctly recognized in the upper part of the oosphere
(figs. 212-215, plate XIX). Dixon ('94) noted the passage into
the oosphere of the four nuclei of the pollen-tube, but he could
not distinguish between these after their entrance into the egg.
Blackman confirmed Dixon's observations as to the passage of
these nuclei into the oosphere and believed that the cytoplasm
114 MARGARET C. FERGUSON
of the " sperm-cells, "passed into the egg along with the sperm-
nuclei but he was unable to demonstrate the fact. There can be
no doubt that the cytoplasm of the sperm-cell enters the egg in
Ftnus (fig. 212). This cytoplasm very soon fuses with that of
the egg and the larger sperm-nucleus moves towards the nucleus
of the oosphere ; the other elements from the pollen-tube remain
for some time in the upper part of the ovum. There is no evi-
dence that the sperm-nucleus increases in size after entering the
oosphere ; neither is their an increase in stainable substance, but,
on the contrary, the nucleus loses its dense structure ; and occa-
sionally a nucleolus becomes apparent within it. (Compare the
sperm-nuclei in figs. 212 and 213 with those in figs. 215-223, «.)
Union of the Sexual Nuclei. — There is every indication that
the movement, within the egg, of the sperm-nucleus which be-
comes active in fertilization is both rapid and direct. It almost
invariably traverses the shortest distance between its point of
entrance into the egg and the egg-nucleus. The relative posi-
tion which the conjugating nuclei may occupy with reference
to the major axis of the oosphere varies considerably, but
always bears a definite relation to the position of the neck cells.
When these cells are directly above the center of the oosphere,
the sperm-nucleus comes into contact with the upper part of the
egg-nucleus (figs. 214, 217, 218, 221, and 223, a) ; but if the
neck be eccentrically placed, the sperm-nucleus will be found
against one side of the oosphere nucleus (figs. 216, 219, and
220). I have not observed the male nucleus beneath the egg-
nucleus as figured by Coulter ('97) in Pinus Laricio. Neither is
there a bulging of the egg-nucleus towards the sperm -nucleus,
nor do the sexual nuclei ever approximate in size as shown in
this same figure of Coulter's, but a somewhat similar figure has
been observed in Pinus Strobus after the first division of the
"segmentation-nucleus." Schaffner ('96 and '97) also notes a
bulging of the nucleus of the oosphere towards the male nucleus
in Alisma and in Sagittaria> but, as will be shown presently,
the exact converse of this is true in the pines which I have
investigated. The sperm-nucleus is usually described as being
more dense than the egg-nucleus at the time of their conjuga-
tion, and I have sometimes found this to be the case in Pinus;
LIFE HISTORY OF PINUS
but as a rule, the conjugation-nuclei in the pines, as observed
by Arnoldi ('oo) in Cephalotaxus, differ in size only (figs.
215-223, a).
Just before the sexual nuclei come into contact, the side of
the egg-nucleus adjacent to the sperm-nucleus becomes slightly
concave (fig. 216). This concavity is doubtless formed under
the influence of the approaching sperm-nucleus and suggests
the crater-like depression developed at an earlier period in the
egg-nucleus of Cycas (Ikeno, '98). As noted by Blackman,
the sperm-nucleus does not penetrate the membrane of the egg-
nucleus, but it lies in a pocket-like indentation formed as a
result of the contact of the two nuclei in the side of the oosphere-
nucleus. Thus both nuclei though still perfectly distinct and
lying side by side, come to occupy the space originally filled
by the egg-nucleus. The sperm-nucleus, when in contact with
the nucleus of the egg, ordinarily assumes the form of a bicon-
vex lens, but it may vary much in outline, presenting in some
cases the figure of a crescent, and in others, that of an ellipse.
Occasionally it forms a deep, tongue-like depression in the
nucleus of the oosphere (figs. 214-223, a).
THE FIRST DIVISION FOLLOWING FECUNDATION.
The Prof hases of the Division. — When the sexual nuclei come
to lie in intimate contact, but are still, to all appearances, per-
fectly distinct, certain changes in their structure indicate that
each is in the early prophase of division. The chromatin con-
denses or collects in irregular granules about the periphery of
the sperm-nucleus, while that of the egg-nucleus is deposited
just beneath the sperm-nucleus. The remainder of each nucleus
is filled with a granular, achromatic reticulum of great beauty,
reminding one of delicate frost work (fig. 224). This condition
suggests an early stage of fertilization in the sea-urchin as de-
scribed by Wilson ('95). Wilson thinks that the sudden increase
in linin may be only apparent, resulting from the " rapid con-
densation and localization of the chromatic substance " ; but he
is inclined to believe that "a considerable portion of the chro-
matin breaks down at this time into linin." It would appear
that the prominence of the achromatic reticulum in the conju-
Il6 MARGARET C. FERGUSON
gating nuclei of Pinus results from both these processes. For,
while there is always a large quantity of linin in the egg-nucleus
and a comparatively small amount of chromatin, the size of the
chromatic spireme, when formed, seems disproportionate to the
entire bulk of the chromatin earlier existing in the nucleus.
The chromatin continues to separate out from these nuclei
until a spireme, studded with irregular granules, lies just within
the wall of the sperm-nucleus, and a similar one arises directly
below in the egg-nucleus. Frequently the cytoplasm caught
between the two nuclei collects into spherical masses ; between
these spheres of cytoplasm the membranes of the two nuclei are
in close contact (fig. 225). Very soon the spireme of each
nucleus becomes coiled and regularly moniliform, and the chro-
matic band of the sperm-nucleus takes up a position along that
side of its nucleus which is nearest to the spireme formed in the
egg-nucleus. At this time, delicate, minutely granular threads,
some of which pass from nucleus to nucleus, appear in the
regions of the two chromatic spiremes. The rest of the achro-
matic contents of these nuclei is largely transformed into long,
comparatively heavy threads, which are furnished with innu-
merable granules. The two nuclei are still perfectly distinct
and the nucleolus of the egg-nucleus may persist at this stage ;
the nuclear membranes are yet present, although they are very
irregular in outline and have given way at several points (fig.
227). The nucleolus is not always present at this time, but
nucleolus-like masses, which from their position are evidently
derived from the egg-nucleus, may be present as late as the
telophase of the division. Delicate, granular fibers continue to
arise in the regions of the two spiremes ; the coarser, achroma-
tic threads of the nuclei become finer in structure, and extend
in all directions toward the forming spindle; and the nuclear
membranes fade entirely out, not only along the line of contact
of the two nuclei, but from their entire outer surfaces as well
(fig. 227). Blackman states that, while the chromatic portions
of these nuclei remain distinct in Pinus sylvestris^ the nuclei
fuse at an early stage in the prophase of the division. There
is, apparently, no such fusion of the sexual nuclei in the species
of pines studied by the writer ; but the entire membrane of each
LIFE HISTORY OF PINUS Il7
nucleus disappears during an early prophase of the mitosis, and
the contents of the nuclei lie free in the cytoplasm of the egg.
I have never found in the process of fertilization in Pinus any
structure that could properly be designated as a fusion-nucleus.
This is exactly comparable with what has been observed in the
ovum of some animals, but has not been previously described
for any plant. It might be noted that this conclusion was
reached very early in the course of these studies, when the
writer had read but little along cytological lines, and was not
aware either that such a process was unknown in plants, or that
a similar conduct of the sexual nuclei had been described by
some writers on the animal side.
As the mitosis proceeds, the spindle-fibers continue to in-
crease in number, becoming even more delicate in structure,
and losing their granular appearance. The long, now quite
delicate, but still granular, achromatic threads of the nuclei are
very numerous, and many extend into the areas occupied by the
chromatic spiremes. They probably feed the growing spindle,
some of them, doubtless, being directly transformed into spindle,
fibers. The chromatic bands have now become perfectly homo-
geneous. Before their segmentation, the very irregular, multi-
polar polyarch spindle has become a multipolar diarch spindle ;
and the achromatic substance not used in spindle-formation
has been gradually resolved, from the periphery of the nucleus
inwards, into a granular, or finely reticulated structure, which
later merges into the general cytoplasm of the egg (figs. 229-
231). When the spindle has become a true multipolar diarch,
it frequently consists of two nearly equal parts, which seem to
belong respectively to the male and female nuclei (figs. 231 and
232, plate XXI). This appearance, however, may be only acci-
dental, as the great irregularity which characterizes this spindle
in the first stages of its formation renders such an origin of the
two halves of the nearly completed spindle very problematic.
Two chromatic groups are distinctly recognized at the time
of the segmentation of the spiremes and can still be clearly made
out during the early development of the chromosomes (figs.
232 and 233). When the chromosomes are being oriented at the
nuclear plate the maternal and paternal elements can no longer
Il8 MARGARET C. FERGUSON
be distinguished (fig. 234). One beautiful preparation was ob
tained at this stage in which a single section through the nuclear
plate showed twenty-four entire chromosomes, and no chromo-
somes were found in the other sections of the series (fig. 235).
As twelve chromosomes had previously been counted in the egg-
nucleus there can be little doubt that the same number is brought
into the egg by the sperm-nucleus. So far as form and struc-
ture are concerned the twenty-four chromosomes of this prepara-
tion are exactly alike, and at this stage I was no longer able
to distinguish between the maternal and the paternal segments.
The smallness of the mitotic figure in the first division fol-
lowing fecundation compared with the size of the egg-nucleus
has been commented upon by Strasburger ('92) and by all later
students of the Abietineas. This spindle may occupy various
positions in the space originally filled by the egg-nucleus, but,
as is clearly demonstrated by a study of its development, it
invariably lies partly within the sperm- and partly within the
egg-nucleus, its major axis being always parallel with the outer,
free surface of the sperm-nucleus. While, then, the karyo-
kinetic figure bears a certain definite, fixed relation to the con-
jugating nuclei, it will be readily seen that its position may
vary, depending upon the shape of the sperm-nucleus and its
line of contact with the egg-nucleus, as, also, upon the plane at
which the section is cut with regard to the sexual nuclei. For
instance, when the sperm-nucleus is elliptical in outline and lies
in a deep depression in the egg-nucleus, as illustrated in figs.
221 and 223, a., plate XX, the spindle will appear to occupy the
center of the egg-nucleus. Cases like the above and many
others were first satisfactorily interpreted after a careful study
of something like two hundred preparations showing fertiliza-
tion stages.
Later Stages in the Mitosis. — During metakinesis the
mitotic figure may present every variation between the ex-
tremely broad, multipolar diarch, shown in fig. 236, and the
narrow, almost bipolar spindle, illustrated in fig. 237. Itas at
this time that the longitudinal splitting of the chromosomes first
becomes apparent. Each chromatic element divides at the point
where the spindle-fibers are attached, forming a small diamond-
LIFE HISTORY OF PINUS 1 19
shaped opening. While this opening is still inconspicuous, the
two halves of a given chromosome become distinct throughout
the entire length of the segment. Such a condition was several
times observed in the division of the " segmentation-nucleus,"
but was not sketched because of lack of space. A similar stage
in the division of one of the four nuclei of the proembryo is
shown in fig. 253, 3, plate XXIII.
In general the chromosomes of the nuclear plate are in the
form of U's and V's ; in rare instances they are long and some-
what coiled, and the spindle-fibers are not attached to their cen-
ters (figs. 234-238). They pass to the poles as narrow U's
(fig. 239). Sometimes the arms of the U are pressed so closely
together that the chromosomes look like longitudinally split
rods. In a late anaphase of the division the chromatic ele-
ments present a crinkled appearance, and the poles of the spin-
dle terminate in granular areas from which threads extend into
the surrounding cytoplasm. These fibers may be quite incon-
spicuous or they may be very prominent, frequently forming
fantastic figures (figs. 240 and 241).
A portion of the achromatic constituents of the sexual nuclei
may persist in the region of the mitotic figure until the forma-
tion of the daughter-nuclei, but, as a rule, all traces of the
original nuclei have disappeared at this time. Blackman finds
no suggestion of a cell-wall in connection with the first division
which takes place within the oosphere. But here, again, I have
found great variation. The spindle either becomes constricted
at the center with little or no sign of thickening along it's median
line, or it may be very broad, in which case prominent thicken-
ings occur, only to disappear at a later stage, in the line of the
cell-plate (figs. 239 and 242). As the half chromosomes unite to
form the daughter-nuclei the poles of the spindle often become
very slender and seem to press against the forming nuclei, ren-
dering them concave along their inner surfaces ; and delicate
fibers now extend from all sides of the division-figure into the
cytoplasm (fig. 242). As already indicated, there is no evi-
dence that any portion of this spindle is derived from the cyto-
plasm, and it is probable that a large part, if not all, of its
fibers are formed by a rearrangement of a portion of the achro-
I2O MARGARET C. FERGUSON
matic, nuclear reticula. During the dissolution of the mitotic
figure some of the substance of the spindle-threads probably
passes into the daughter-nuclei, but the greater part of the
fibers merge into the cytoplasmic reticulum and become indis-
tinguishable from it. We have here another evidence that
cytoplasmic and nuclear elements are but different expressions
of the fundamental or ground substance of the cell. When the
daughter-nuclei are formed they present very beautiful, monili-
form reticula, which later undergo changes very similar to
those described for the growing egg-nucleus.
As recorded by Wilson ('96 and 'oo), Van Beneden ('83 and
'87) made the very interesting discovery, later confirmed by
Herla ('93) that the chromosomes are formed separately in the
sexual nuclei of Ascaris megalocephala. The differentiation
of the chromatic segments takes place after the entrance of the
sperm-nucleus into the egg but before the two nuclei have come
into contact. Thus the exact equivalence of the chomatic sub-
stance in the paternal or maternal nuclei was demonstrated. In
the following year, Strasburger ('88) suggested that in the com-
ing together of the nuclear theads lay the important point in
fertilization. A separating-out of the chromatic elements simi-
lar to that described by Van Beneden, has since been found to
occur during fertilization in many animals, but has not yet been
demonstrated as of frequent occurrence in plants. In 1891,
Guignard described the formation of two distinct chromatic
spir ernes in the copulation nucleus of Lilium Martagon, but
he did not figure them, and his statement seems to have been
overlooked by most later writers. Strasburger was able, in
1897, to distinguish the maternal and paternal portions of the
fertilized nucleus in Fucus up to the time when the spindle was
fully formed, and Ikeda ('02) states, regarding Trycirtis:
"The paternal and maternal chromatin elements of the result-
ing nucleus are distinguishable long after fusion." But the
results of more recent writers T seem to indicate that fertilization
1 Arnoldi ('oo) in Cephalotaxus, and ('01) in Sequoia' Caldwell ('99) in
Lemna ; Campbell ('99) in Spharganium ; Farmer and Williams ('98) in Fucus ;
Guignard ('99) in Lilium j Harper ('oo) in Pyronema ; Ikeno ('98) in Cycas
and ('01) in Ginkgo ; Jager ('99) in Taxus ; Land ('oo) in Erigeron and Sil-
phium : Lotsy ('99) in Gnetum ; Merrell ('oo) in Silphium ; Mijake ('01) in Pyth-
LIFE HISTORY OF PINUS 121
in plants consists in the fusion of the two nuclei to form a rest-
ing nucleus not demonstrably different, except in some cases in
its greater size, from the original egg-nucleus.
Students of certain of the Abietinece, however, have attained
quite different results, and find in these plants phenomena very
similar to those occurring during fertilization in some animals.
Blackman concludes that in Pinus sylvestris " no resting fertil-
ized nucleus is ever formed" and that "the half-chromosomes
derived from the male and female nuclei respectively, fuse
together at the poles of the first segmentation spindle " ; and
Chamberlain found that two chromatic spiremes were formed in
Pinus LariciO) but, as so many stages were lacking in his
material, he hesitated to draw definite conclusions ; Woycicki
('99) reported a complete fusion of the sexual nuclei in Larioc^
but in some cases he saw two chromatin-groups, and suggested
that they might have been derived, one from each parent ; and
Murrill ('oo) has recently described the formation of two distinct
spiremes in Tsuga. As a result of the present studies, it has
been shown conclusively, as stated by the writer in i9Oilands,
that the chromatic portions of the sexual nuclei remain distinct
until the daughter-nuclei are formed ; and there is, moreover,
never any true fusion of the conjugating nuclei, that is, the two
nuclei do not form one individual enclosed by a definite
membrane.
It is evident from the foregoing, that fertilization in Pinus
consists in the complete union of two cells. Cytoplasm fuses
with cytoplasm and nucleus unites with nucleus.
No centrosome or centrosome-like body has been observed in
connection with the sexual nuclei, either before or during this
division. Although the centrosome as an organ has failed to
be demonstrated, yet a detailed study of this mitosis makes the
conclusion inevitable that the force initiating and controlling
the division is supplied by the sperm- and not by the egg-nucleus
turn; Mottier ('98) in Lilium and ('oo) in Dictyota; Nawaschin ('99) in Lilium,
and ('oo) in Helianthus, Delphinium and Rudbeckia; Osterhaut ('oo) in Batra-
chospermum ; Shaw ('98) in Onoclea ; Thorn ('99) in Adiantum and Aspidium /
Thomas ('oo) in Caltha ; Wager ('oo) in Peronospora ; Webber ('01) in Zamia ;
all who have described coiled sperm-nuclei ; and all writers with the exception
of Ikeda who have published on fertilization in plants during 1902 and 1903.
Proc. Wash. Acad. Sci., August, 1904.
T22 MARGARET C. FERGUSON
— this force manifesting itself only in the presence of the egg-
cytoplasm.
The demonstration of normal parthenogenesis in several
plants and of artificial parthenogenesis by Nathanson ('oo) has
led to much interesting discussion regarding the nature of the
stimulus exerted by the sperm on the egg. Klebs ('01) sug-
gests that it is merely of the nature of an external shock, and
other explanations have been offered ; but, after carefully
reviewing the literature of the subject, Zacharias ('01) con-
cludes that we have still to determine the true nature of the
stimulus which the sperm exercises upon the egg, and in so far
as I am aware, none of the more recent studies have thrown
any substantial light on this problem.
Nothing has been observed throughout this study to indicate
that the sperm-nuclei of Pinus ever assume the spiral or reni-
form shape, suggestive of spermatozoids, which has been
described by recent writers l for the sperm-nuclei in various
Phanerogams, and by Arnoldi ('01) in Taxodium and Sequoia.
But the nuclei early become spherical or elliptical in outline,
depending on the breadth of the pollen-tube, and remain so dur-
ing their entire later history.
THE PRO-EMBRYO.
Division oftheTiuo Segmentation-nuclei. — The two daughter-
nuclei remain in the upper part of the egg and pass through
the same stages in their development as those described in the
maturation of the egg-nucleus, except that, as a rule, no nu-
cleolus becomes apparent within them. These nuclei have
been observed to approximate in size the mature egg-nucleus ;
but they usually cease to grow and begin to divide while they
are still much smaller than the fully developed nucleus of the
oosphere. The steps in the division of these two nuclei in
Pinus Strobus^ this division has not been carefully studied in
the other species, are almost exactly like those of the first divi-
vision. The nuclear reticulum is resolved into a beautiful, open
1Golinski ('93) in certain grasses ; Nawaschin ('98), Guignard ('99), Sargant
('99) in Lilium; Guignard ('oo) in Tulip a / Land ('oo) in Composites; Merrell
('oo) in Silphium; Strasburger ('oo) in Monotropa; Thomas ('oo) in Caltha ;
and many others during the past two years.
LIFE HISTORY OF PINUS 123
and interrupted, granular, achromatic network which is crossed
by several coarsely granular, deeply staining threads. These
threads, which represent the chromatic portion of the nucleus,
have at first no definite arrangement ; but they soon unite to
form two distinct, coiled or angled spiremes, which draw to-
gether at one side of the nucleus (figs. 244, 245). It is an
interesting fact that these spiremes are always found on adja-
cent sides of the two nuclei. This position suggests that there
is a certain attraction, comparable to that existing between the
sexual nuclei, active between these nuclei ; or the relation of
the inner sides of these nuclei with the poles of the spindle, in
the early stages of their formation, may have some influence
upon the position which these spiremes assume in the dividing
nuclei.
When the two spiremes, which are still roughly beaded with
the chromatic substance, come to lie side by side along the inner
wall of the nucleus, the nuclear wall resolves itself into a weft
of fibers. These threads pass into the surrounding cytoplasm
and soon wholly disappear, while, at the same time, achromatic
fibers arise in the regions of the spiremes (fig. 245). The
achromatic threads quickly draw together, forming a sharply
bipolar spindle on which the two now perfectly homogeneous
chromatic bands lie. The spindle does not become bipolar in
some instances until after the segmentation of the spiremes
(figs. 245-247). I have preparations representing a complete
series in this division, but, as it is exactly similar, especially in
its later stages, to the first division, it is not thought best to
multiply sketches by repeating like figures.
There can be little doubt that the two spiremes formed in each
of these nuclei represent the separated-out paternal and maternal
chromatic substance, although to all appearances, the chromo-
somes were completely fused in the reticula of the daughter-
nuclei. One is reminded by these phenomena, of Strasburger's
('92) remark, when he states that he accepts the view of a com-
plete fusion of the segments into a network in the daughter-
nuclei, and then asks if he must, therefore, conclude that the
chromosomes in the following divisions do not correspond in
material. This restoration of the paternal and the maternal
124 MARGARET C. FERGUSON
chromatin from a finely divided network is certainly strongly in
favor of the theory of the individuality of the chromosomes ; and
it is this phenomenon, noted many months before microsporo-
genesis was carefully studied, together with the method of the
origin of the chromosomes in the first and second divisions of the
microspore-mother-cell, that inclines me to accept the view that
the chromosomes in the homotypical division of the microspore-
mother-cell are identical with those formed in the metaphase of
the heterotypical mitosis.1 Moreover, this phenomenon, here
observed for the first time in plants, would seem to add substan-
tial interest from a cytological point of view to Mendel's laws
which are at present being so ardently discussed both by animal-
and by plant-breeders.
Riickert ('95) found that the chromatic portions of the conju-
gating nuclei in Cyclops not only remain distinct during the
first division, but the two groups of chromosomes, representing
respectively the maternal and the paternal chromatic elements,
could still be recognized after several divisions had taken place.
In this case, however, the two groups do not fuse in the daughter-
nuclei but a double nucleus is formed in the resting stage. In
the same year Zoja ('95) observed that in Ascaris the maternal and
the paternal chromosomes remain entirely distinct during several
successive divisions of the segmentation nucleus. We have,
then, in this second division a further point in which fertiliza-
tion-phenomena in Pinus correspond to those which occur within
the ova of some animals. I have, as yet, made no attempt to
obtain a complete series of stages in the development subsequent
to the formation of the first four nuclei of the proembryo. But,
from a comparison of fig. 252, ft, plate XXIII, with 244, plate
XXII, and 253, b, plate XXIII, with 237, plate XXI, one is led
to expect that the third division following fertilization will corre-
spond in all points with the second. It would be interesting to
determine if two chromatic groups are characteristic of all
divisions which normally occur within the oosphere of Pinus,
and I hope to investigate this question more thoroughly at some
future time.
The Four Segmentation Nuclei. — As a rule, these nuclei
1 See note at close of Appendix.
LIFE HISTORY OF PINUS 125
retain their position in the upper half of the egg until their
growth is completed (fig. 249). Here, again, as in the develop-
ment of the two segmentation nuclei, the steps described for the
maturation of the egg-nucleus are repeated, except that a
nucleolus does not generally become apparent within these
nuclei. After attaining full size, the four nuclei pass to the base
of the oosphere, as described by all recent writers. During their
descent many fibers arise in the cytoplasm surrounding the
nuclei. Some of these threads run parallel with the walls of
the nuclei, while others extend out from the nuclei in a radial
manner. These fibers become more prominent as the nuclei
approach the base of the oosphere, and, as in the case of the
egg-nucleus, they are most strongly developed along the upper
sides of the nuclei (figs. 250, #-251, b}. Blackman suggests a
relation between these fibers and the walls that arise later at
the organic apex of the oosphere, but I find no evidence of
any connection between the two. When these nuclei have
nearly reached the bottom of the egg, the nutritive spheres
have almost disappeared from the cytoplasm, those which
still persist being much reduced in contents (fig. 251, a).
After the four nuclei have arranged themselves at the " organic
apex " of the oosphere, in a plane perpendicular to the major
axis of the archegonium, a marked change occurs in the cyto-
plasm of their immediate vicinity. It becomes dense, coarse,
more or less granular, and has a great affinity for stains (figs.
252, a and 3, plate XXIII).
The early prophases, as also the meta- and anaphases in the
mitosis of the four segmentation nuclei, in so far as studied,
correspond in every respect with the same stages in the second
division following fertilization ; and it is probable that the
chromosomes are derived from two distinct spiremes as in the
first and second divisions occurring within the egg ; but, as
already indicated, the steps in the origin and development of
the chromosomes have not been carefully traced in this division.
These nuclei divide simultaneously. Chamberlain states that
"in the division of the four nuclei the spindle is extremely
broad and multipolar." I have occasionally observed such a
figure during this mitosis, but here, again, great variation exists.
126 MARGARET C. FERGUSON
Every transitional form may be presented during the metakinesis
between a multipolar diarch spindle, which fills the entire breadth
of the nucleus, and a slender bipolar spindle, such as is shown
in fig. 253, b. As the halves of each chromosome separate at
the point where the spindle-fibers are attached, the longitudinal
splitting of the segments becomes evident throughout the entire
length of the chromosomes (fig. 253, b.)
The Development of Cell-walls. — During mitosis, the deeply
staining substance surrounding these nuclei condenses into large
irregular masses at the periphery of the nucleus. When the
eight nuclei are formed this deeply staining material collects
about them and extends in irregular strands into the cytoplasm.
Each nucleus is now surrounded by its own cytoplasm, though
no cell-walls have yet been laid down (figs. 253, £, and 254, b).
Blackman describes the formation of cell-walls about the four
nuclei at the base of the archegonium, and Coulter and Cham-
berlain state that cross-walls separating these nuclei, but leav-
ing them exposed above, arise when the four nuclei have
arranged themselves at the base of the oosphere, and are under-
going division. In the five species of pines which I have studied
cell-walls do not arise until after eight nuclei have been formed.
The deeply staining cytoplasmic substance appears to be
repelled from all sides of these nuclei and is deposited in lines
which indicate the position of the future cell-walls ; the cell-
membranes appear to arise by a direct transformation of this sub-
stance. The process seems to be very similar to that described
by Farmer and Williams ('98) in Fucus. Mottier (Joo) inclines
to the view that the cell-plate is deposited in the form of a homo-
geneous fluid, the kinoplasm, even though its presence cannot
be demonstrated, being the active agent in its deposition. The
substance which is cast out, or passes but, from the region of
the eight nuclei in the formation of cell-walls at the base of the
oosphere in Ptnus, has the appearance at times of a homogene-
ous, deeply staining fluid, in which numerous irregular granules
are imbedded ; but there is never any evidence of its being purely
fluid in nature. It seems very probable that the large granules
cast out from the cytoplasm surrounding these nuclei at this
time are similar to the smaller granules deposited at the cell-
LIFE HISTORY OF PINUS 127
plate during the ordinary process of cell-wall formation. That
the granules are larger and the details of the process more
striking here may be accounted for by the fact that, under the
influence of each nucleus, three times as much cell-wall must
be laid down as is ordinarily formed by the action of a single
nucleus. But in any case, we are still far from a satisfactory
understanding of the method by which cell-walls arise.
The eight nuclei are arranged, as usually described, in two
tiers of four cells each. The cytoplasm of the upper four cells
remains continuous with the cytoplasm of the egg, that is, a
dividing wall is not formed along their upper surface (figs.
255, a, and 255, b).
Later Mitoses in the Formation of the Proembryo. — The
second set of division figures which occurs at the organic apex
of the egg arises in the upper tier of cells, that is, in the four
cells which have never been cut off from the general cyto-
plasm of the egg (fig. 256). This is contrary to all reports
of the development of the proembryo in the Abietinea.1 The
second division occurring in the nuclei at the base of the
archegonium has not been previously observed, so far as I
am aware, and, the third division occurring in the basal tier
of cells, the inference seems to have been made that the cells
which are not enclosed along their upper sides by definite
walls never divide. Coulter and Chamberlain ('01) make the
remark that the upper four free nuclei increase much in size,
and they figure them in the spireme stage ; but they do not refer
to the fact that they are in the prophaseof division, and describe
all further mitoses after the eight-celled stage as occurring in
the basal tier of cells. Strasburger and Hillhouse ('oo) also
describe the further development of the proembryo in Picea
after the establishment of cell-walls, as proceeding from two
successive divisions of the four basal cells.
It seems to me a rather significant fact that the four cells
which remain in open communication with the egg should not
only divide again, but that their division should be entirely
completed before the cells of the lower tier show any signs of
dividing. There are thus, in Pinus, four successive mitoses
1 See note at close of Appendix.
128 MARGARET C. FERGUSON
resulting in the formation of twelve nuclei under the direct in-
fluence of the egg-cytoplasm, rather than three divisions with
the formation of eight nuclei as has been previously described.
The phylogenetic bearing of this phenomenon may be more
far-reaching than is at first apparent, suggesting as it does a
possible closer relationship with those lower gymnosperms in
which many free nuclei arise in the egg before the deposition
of cell-walls.
At present I can give only this general outline of the origin
of the proembryo, but I hope to be able in the near future to
make a detailed study of the several mitoses which occur here
in Pinus.
The Fate Within the Egg of the Smaller Sperm-nucleus ,
the Stalk-cell^ and the Tube-nucleus. — When the various ele-
ments from the male gametophyte first enter the oosphere, there
is no question as to the identity of the several nuclei to one who
has become familiar with them before their exit from the pollen-
tube (figs. 213-215, plate XIX). Remnants of these cells have
been found in the upper part of the egg as late as the forma-
tion of the eight-celled stage of the proembryo. The stalk-
cell remains for some time unchanged and finally disintegrates.
In so far as I have been able to determine, it assumes a more
or less granular appearance, and at last blends with the cyto-
plasm of the egg. The tube-nucleus undergoes various changes.
Occasionally it seems to contract, becoming gradually smaller
until it is no longer demonstrable ; it may change little, if any,
in size, but its reticulum often becomes more prominent than
when within the pollen tube ; rarely it enlarges rapidly after its
entrance into the egg and develops a beautiful reticulum (fig.
212, plate XIX). The sperm-nucleus not active in fertilization
increases but little in size, and its network becomes less dense,
resembling that of the conjugating nuclei ; it may pass through
the ordinary processes of disintegration ; and in a few cases it
has been observed to divide amitotically, as described by Ar-
noldi ('GO) in Cephalotaxus.
But frequently the sperm-nucleus and occasionally the tube-
nucleus attempt to divide mitotically. One or two small, abor-
tive, karyokinetic figures are not uncommon in the upper part
LIFE HISTORY OF PINUS 1 29
of the egg at the time of the division of the two segmentation-
nuclei. I have said " attempt to divide," for no instance has
been observed in which the division of these nuclei has extended
beyond a late prophase. A bipolar spindle, with the chromatic
segments scattered irregularly upon it, represents the most ad-
vanced stage which has been seen in the division of the smaller
sperm-nucleus (fig. 259, £, plate XXIII). (During sectioning, a
rupture was made in the cytoplasm at one end of this spindle
so that the upper pole has been separated into two.) The stalk-
cell still persists at this late date (fig. 259, 3, plate XXIII),
and in another section of the series (fig. 259, a\ a second
mitotic figure appears. This evidently represents the tube-
nucleus. The achromatic part of the figure presents the ap-
pearance of a normal bipolar spindle, but, the chromatic spireme
has not become homogeneous and probably would not have
developed further. In some cases a well-developed spireme is
formed in the upper part of the egg, but no achromatic threads
become apparent (fig. 257); again, a nucleus seems to have
been entirely resolved, during its disintegration, into achromatic
fibers. As above stated, in no case observed did the division
of these nuclei reach telekinesis, but at some point in the devel-
opment prior to such a late stage, activity ceased and disinte-
gration of the nuclear elements took place. Murrill ('oo) ob-
served a similar figure, which he interpreted as the smaller
sperm-nucleus, in the upper part of the fertilized egg in Tsuga.1
It might be suggested that these division-figures result from
the conjugation of the nucleus of the ventral canal-cell with the
smaller sperm-nucleus. There is no evidence that such is the
case, and I am convinced that they could not have had such an
origin. In an examination of many hundred archegonia just
before fertilization, but one ventral canal-cell containing a nor-
mal nucleus has been observed. Shall we, then, conclude that,
in a far less number of preparations representing stages imme-
diately following fecundation, fifty or more instances occur in
which the nucleus of the ventral canal-cell has conjugated with
another nucleus and subsequently divided?
It is generally recognized, especially by cytologists on the
1 See note at close of Appendix.
I3O MARGARET C. FERGUSON
animal side, that the stimulus to division is given not by the egg-
nucleus, but by the cytoplasm of the egg. If this be true, it is
not strange that these nuclei, lying in a position where everything
is most favorable for growth and development — in a medium
not only rich in nutritive substances but especially adapted to
incite activity in nuclei — should divide. It is a well-known
fact that when several spermatozoa enter the ovum of certain
animals, only one unites with the egg-nucleus, the others de-
generate, or, as is frequently the case, they divide mitotically.
And herein we find a further similarity between the processes
attending fertilization in some animals, and those taking place
within the oosphere of Pmus.
SUMMARY.
At the time of fertilization, an opening is formed in the apex
of the pollen-tube, and the cells of the male gametophyte which
still persist, together with a portion of the cytoplasm and some
of the starch of the pollen-tube, pass into the cytoplasm of the
egg-
The larger sperm-nucleus escapes from the protoplasm of the
sperm-cell and moves directly toward the egg-nucleus ; the
other nuclei from the pollen-tube may persist, in a modified
form, in the upper part of the archegonium until the eight-
celled stage of the proembryo ; but the cytoplasm of the sperm-
cell soon fuses with that of the oosphere. The stalk-cell grad-
ually disintegrates and blends with the egg-cytoplasm. The
tube-nucleus and the smaller sperm-nucleus may share the fate
of the stalk-cell, but, during the second division of the egg, they
not frequently give rise to mitotic figures. The smaller sperm-
nucleus, then, may pass through a slow process of disintegration,
it may divide amitotically, or it may give rise to a karyokinetic
figure of more or less definiteness.
There is no apparent change in the diameter of the sperm-
nucleus after its entrance into the oosphere. At the time of
conjugation, the egg-nucleus is several times larger than the
sperm-nucleus, and the sperm-nucleus does not increase in size
after its contact with the egg-nucleus. The inequality in size
of the sexual nuclei may be due to the difference in the size of
LIFE HISTORY OF PINUS
their cells. But if, as has been suggested, the egg-nucleus
functions as a manufacturer of nutritive material, may we not
find in this activity a feasible explanation of its greater size ?
The conjugating nuclei, always dissimilar in size, may or may
not be dissimilar in structure.
The egg-nucleus becomes slightly convave on the side nearest
to the approaching sperm-nucleus. This nucleus imbeds itself
in the side of the egg-nucleus but does not penetrate its mem-
brane. A chromatic spireme arises, and a prominent achroma-
tic reticulum becomes apparent in each nucleus. Soon after-
wards the nuclear membranes entirely disappear. The two
chromatic groups remain distinct until the nuclear plate stage.
Fertilization consist in Pinus in the union of two entire cells.
Cytoplasm fuses with cytoplasm, but there is never any fusion,
as ordinarily understood, of the sexual nuclei.
The spindle of the first division following fecundation always
lies between the conjugating nuclei and parallel with the outer,
free surface of the sperm-nucleus. It is multipolar in origin
and is probably derived equally from the paternal and the ma-
ternal nucleus. The spindle-fibers appear to arise by a rear-
rangement of the achromatic nuclear reticula and are evidently
not the expression of a special kinoplasmic substance. After
the formation of the daughter-nuclei, the greater portion, if not
all, of these threads pass into the cytoplasmic network. Dur-
ing metakinesis and later stages this spindle may vary from a
broad, multipolar diarch to a slender, bipolar spindle. The
chromosomes pass to the poles in the form of narrow U's.
No individualized centrosomes or centrospheres have been
found to occur in connection with the first division following fer-
tilization. But the entire activity connected with this mitosis indi-
cates that the sperm-nucleus acting in the presence of the egg-cy-
toplasm is the agent which initiates and controls the division.
The two segmentation-nuclei present a reticulated structure
in which the paternal and the maternal chromatin appear to be
completely fused. They divide in the upper part of the egg
passing through practically the same steps as those noted for
the first division. The two chromatic spiremes formed in each
nucleus take up a position along the adjacent sides of the
13 2 MARGARET C. FERGUSON
nuclei. These bands without doubt represent the separated-out
paternal and maternal chromatic substance. This phenomenon
is of especial interest in that it suggests a cytological basis for
Mendel's laws. A longitudinal splitting of the chromosomes
first becomes apparent during an early stage in metakinesis.
The four segmentation-nuclei attain full size while still in
the upper part of the egg. As they pass to the base of the
oosphere, fibers occur in the cytoplasm similar to the threads
observed around the growing egg-nucleus. The steps in the
division of these nuclei have not been carefully traced, but,
from the stages observed, it is probable that this mitosis does
not differ from the division of the two segmentation-nuclei.
No cell-wall is laid down at the base of the oosphere until
after the eight-celled stage of the proembryo has been reached.
These eight nuclei are surrounded by a deeply-staining granu-
lar substance which extends out from each nucleus in irregular
strands. This substance finally comes to lie in the lines of the
future cell-walls and is evidently transformed into cell-wall.
It is probably not different from the smaller granules deposited
in the line of the cell-plate during the accustomed method of
cell-wall formation.
The fourth division which occurs within the fertilized egg
takes place in the four cells of the upper tier of cells at the
base of the archegonium. Thus twelve nuclei are formed
under the direct influence of the egg-cytoplasm. This fact
herein noted for the first time1 is significant, suggesting as
is does a closer relationship with those lower gymnosperms in
which many nuclei are formed in the cytoplasm of the egg.
The number of chromosomes in the nucleus of the ventral
canal-cell, in the nuclei of the sheath-cells, and in the egg-
nucleus has been found to be twelve, while the mitotic figure,
in the first division following fertilization, shows twenty-four
chromatic segments.
It is interesting to note the many points of similarity between
fertilization as it has been observed in Pinus, and the processes
known to take place during fertilization in some animals, (i)
The egg in Pinus is very large and is abundantly supplied with
1 See note at close of Appendix.
LIFE HISTORY OF PINUS 133
nutritive spheres. (2) The sexual nuclei do not fuse, and no
structure which could properly be called a segmentation-nucleus
is ever formed. (3) An achromatic nuclear reticulum becomes
very prominent in the sexual nuclei during the prophase of
division. (4) The chromatin of the sexual nuclei forms two
definite groups which remain distinct until metakinesis. (5)
Two chromatic groups, doubtless representing respectively the
paternal and the maternal chromatin, appear in the second
division following fecundation ; and the indications are that
they will again occur in the third division, and perhaps are
characteristic of all the mitoses which take place within the
oosphere. (6) The nuclei, which enter the egg but play no
part in fertilization, show a tendency to divide mitotically.
The conclusions reached throughout this paper hold good,
when not otherwise indicated, for all five species of pines which
I have studied. Nuclear phenomena are found to vary so much,
even within the limits of a given genus, that it is no longer safe
to consider the details of development in a single plant as typical
of a large group of plants. We therefore make no generaliza-
tions regarding the Abietinece. And we hesitate, even, to draw
conclusions for the genus Pinus, for, while the agreement in
certain phases of development of five species would seem to be
sufficient for the formulation of a rule, there may still exist
within the genus individuals which differ, in certain aspects of
their nuclear activity, from that which has been found to occur
in Pinus Strobus, P. austriaca, P. rigida, P. resmosa and P.
montana var. uncmata.
APPENDIX.
SOME ABNORMAL CONDITIONS.
Supernumerary Nuclei in the Male Gametophyte. — Cham-
berlain ('97) described a multiplication of the normal number of
cells in the pollen-grain of Lilium; Arnoldi ('oo) finding more
than the usual number of nuclei in the pollen-tube of Cephalo-
taxus, considered that more than one tube-nucleus had been
formed ; and Coker ('02) has very recently found that both the
first and second prothallial cells in Podocarpus may divide
134 MARGARET C. FERGUSON
mitotically. I have only three times observed an excess of the
normal number of nuclei in the male gametophyte of Pinus.
Three nuclei have been found in the pollen-grain after the tube-
nucleus has passed into the pollen-tube (fig. 271, plate XXIV).
Two nuclei have twice been seen just passing into the pollen-
tube, while the stalk-cell could still be detected in the lower
part of the pollen-grain in one instance, and in the other it had
just left the grain but had not as yet passed the generative cell.
In the former instance (fig. 272) the stalk-cell was almost
obscured by the dead nucellar tissue and is not shown in the
sketch. Here the two nuclei are in close contact, the smaller
nucleus being imbedded in one side of the larger nucleus. In
the second case (fig. 273) the nuclei are farther removed from
the pollen-grain, although still connected with it by the cyto-
plasm of the larger cell ; the smaller nucleus is surrounded by
its own cytoplasm and is in contact with the lower part of the
larger cell.
Any interpretation of these irregularities must be more or less
hypothetical, and yet from the position, size, and structure of
the nuclei certain inferences can be made regarding them. In
the condition represented in fig. 271, one of the prothallial cells
may have persisted, the stalk-cell may have divided, or the
generative cell may have given rise to the extra nucleus. But
considering the character of the nucleus and also that of the
nucleus of the stalk-cell, it seems to me most probable that two
stalk-cells have been formed. In figs. 272 and 273 the proba-
bilities are very strong that the smaller nucleus in each instance
was cut off from the generative nucleus. The stalk-cell is per-
fectly normal in appearance and gives no evidence that it has
passed through any unusual history. The two large nuclei
shown in fig. 273 bear a very striking resemblance to the
sperm-nuclei, and when first observed with a lower power of
the microscope the impression was that the generative nucleus
had divided very early and the smaller sperm-nucleus was in
advance. But, when the higher magnification revealed the
stalk-cell still above these nuclei, and also disclosed the fact that
there were in reality two cells, it at once became apparent that
these are not to be considered sperm-nuclei. For two sperm-
LIFE HISTORY OF PINUS 135
cells are not formed ; the smaller sperm-nucleus is never in ad-
vance ; the generative cell does not give rise to the binucleated
sperm-cell until after the stalk-cell has passed beyond it, nor has
its normal division ever been observed to occur while it is still
united with the pollen-grain by its own cytoplasm. In this case
it seems very evident, then, that two generative cells have arisen
by the division of the first generative cell. Whether both of
these would have divided to produce four sperm-nuclei is of
course a mere matter of conjecture, but the cytoplasm of the
smaller cell is very scanty and it is probable that only the larger
one would have functioned as the generator of the sperm-nuclei.
The uncertainty as to the origin and fate of these extra nuclei is
in each instance too obscure to admit of any theorizing regard-
ing their significance.
Usual Conditions in the Female Gametophyte. — In only one in-
stance has more than one macrospore-mother-cell been observed.
In this case two cells which are very similar and centrally placed
in the spongy tissue differ from the surrounding cells in exactly
the same way as has been described for the young macrospore-
mother-cell (fig. 260, plate XXIII). Farmer ('92) records the
discovery of a double prothallium in Pimis sylvestris, and Hof-
meister had previously made a like observation in the same
species. I find no other instance recorded for Pinus in which
more than one macrospore must have been functional. Shaw
('98) and Arnoldi ('99) find one or more macrospore-mother-cells
in Sequoia from which several embryo-sacs may arise ; Arnoldi
('oo) has made a similar observation for Cunninghamia^ Sciad-
opitys, Taxodium, and Cryptomerta; Lotsy (?992) and others
find many young embryo-sacs in Gnettim; and Coker reports
the prescence of two prothallia in Prodocarpus and Taxodium.
The presence of a multicellular sporogenous tissue has been
reported in the Angiosperms by several students — Nawaschin
(*992) in Corylus, Lloyd ('01) in the Rtibiacea, Murbeck ('01)
in the Rosacece, and by others. The appearance of more than
one functional spore within the ovule of such widely-separated
plants makes it rather doubtful if this character is important
phylogenetically.
Juel ('oo) found that the walls separating the macrospores in
136 MARGARET C. FERGUSON
Larix are often oblique. Only one such instance has been
observed in Pinus and is shown in fig. 261, plate XXIII.
Considerable variation has been noted in the origin of the
archegonia, a few of the irregularities, which are in fact typical
of all, have been figured. Figs. 262, a and b, represent two
sections through the upper part of the same prothallium. They
show twenty-three young archegonia in various stages of devel-
opment. Only a single archegonium of those shown in the
illustrations had its origin in a superficial cell ; some of them
originated in the sheath-cells of normal archegonia found
in other sections, but this fact is not demonstrated in the
sketches; however, in fig. 265, taken from another prothal-
lium, a little archegonium is seen budding, as it were, from
a sheath-cell of the larger archegonium, and in fig. 266
is shown a somewhat similar case except that here one arche-
gonium is directly above the other.1 One would consider
it very doubtful if such an archegonium as this lower one
would develop further; but fig. 267 shows an archegonium
similarly located in which the central cell has divided, and both
the ventral canal-cell and the egg-nucleus are still clearly visi-
ble, though the latter shows some signs of disintegration. In
all these archegonia no neck cells have been formed.
In one instance, nine archegonia were found Pinus in montana
uncinata, so arranged along the top and side of the prothallium
as to suggest a cock's comb — seven of the archegonia being
apparent in a single section. The figure was reconstructed
from several sections in the series and the archegonia overlap
not all lying in the same plane, but they are all plump and
normal though some show early stages in disintegration. The
two at the top have well developed proembryos, but none of
the others have been fertilized (fig. 260). Archegonia are fre-
quently found arranged vertically as in fig. 261. In such cases
as this the lower ones do not arise from the one just above, but
each is connected with the exterior by means of a funnel-shaped
opening leading from its neck-cells to the side of the prothal-
lium ; this cannot be shown in a sketch as it is not evidenced in
any one section, and can only be determined by carefully
studying the whole series.
1 See note at close of Appendix.
LIFE HISTORY OF PINUS 137
It has been held by various students that all the nuclei in the
embryo-sac of Angiosperms are potential eggs. Murbeck ('01)
has recently, as recorded by Overton ('02), demonstrated the
development of an embryo inAlckemella; Chamberlain ('95) dis-
covered the presence of an antipodal oosphere in Aster ; -and
many earlier investigators have made similar observations regard-
ing the synergids and antipodals.1 The discovery of archegonia
that have originated not only from superficial cells at the top and
along the sides of the prothallium, but from cells considerably
removed from the surface as well would seem to give direct
affirmation to the suggestion made by Atkinson ('01) that all the
cells of the prothallium in Gymnosperms are potential eggs.
Among the many archegonia studied, I have found two in
which the nucleus of the ventral canal-cell approximated that
of the egg in size. Fig. 268 shows such a condition in Pinus
Strobus, but even here the nucleus of the ventral canal-cell is
much smaller than that of the egg. It is, however, remarkably
large for the nucleus of the canal-cell in this species, and is
apparently still in a normal condition, whereas this nucleus is
ordinarilly in an advanced stage of disintegration when the egg
has reached maturity. A much more marked increase in the
size of the nucleus of the canal-cell has been observed in Pinus
austriaca as illustrated in fig. 269. Here it has attained a com-
paratively enormous size and presents almost exactly the same
structure as the nucleus of the fully developed egg, though
slightly smaller than the egg-nucleus. Chamberlain ('99) fig-
ures a similar enlargement of the nucleus of the ventral canal-
cell in Pinus Laricio and concludes that this cell is the homo-
logue of the egg. It will be noted that in the instances described
above, no ventral canal-cell has been formed, but that in both
cases the nucleus of the canal-cell lies free in the cytoplasm of
the egg2 (figs. 268, 269). The failure to form a wall cutting
off the ventral canal-cell from the egg, or the early absorption
of this wall if it has been formed, seems to me ample reason
for the unusual size and persistence of the nucleus of the canal-
1 Miss Opperman, a student in my own laboratory, has recently discovered the
fertilization of an antipodal egg in Aster, a description of which is soon to be
published.2
2 See note at close of Appendix.
Proc. Wash. Acad. Sci., September, 1904.
138 MARGARET C. FERGUSON
cell, since it lies in the cytoplasm of a cell which supplies the
most favorable medium for growth found in the plant. Not the
slightest evidence has been observed during this research that
the nucleus of the ventral canal-cell ever divides or that it ever
conjugates either with the egg-nucleus or with the smaller
sperm-nucleus. The fact that this nucleus enlarges when fed
by the cytoplasm of the egg does not seem to me conclusive
evidence that it has been " organized as an egg," as stated by
Coulter and Chamberlain ('01). The tube-nucleus and the
smaller sperm-nucleus often enlarge after their entrance into
the egg but, surely, they are not thereby changed into eggs.
The fragmentation of the egg-nucleus has been observed sev-
eral times and is illustrated in fig. 270. The ventral canal-cell
can still be seen just above the egg. Such fragmentation of the
egg-nucleus is not rare in the Gymnosperms having been re-
ported by various writers.
In one instance one of the two segementation-nuclei was
found to have divided while the other remained undivided. The
undivided nucleus had increased much in size and contained
seven large, granular spheres distributed on an achromatic re-
ticulum. The nucleus is evidently in a state of disintegration
and these spheres probably represent granular masses of chro-
matin (fig. 274).
A Peculiar Method of Conjugation. — Of all the irregular or
abnormal developments observed that illustrated in fig. 275 is,
to me, the most interesting. A pollen-tube has conjugated with
an egg, not through the normal passage formed by the neck-
cells, but has forced its way through the sheath-cells at one
side of the archegonium. Impregnation has evidently followed
and division has taken place as usual, four nuclei of the pro-
embryo having been formed.
The fifth large nucleus shown within the egg is doubtless the
smaller sperm-nucleus. The open space separating the upper
part of the prothallium from the nucellar cap has evidently not
arisen as a result of shrinkage during fixation. The pollen-
tube unable to span the opening has turned aside and finding a
point at which the endosperm and nucellus were in contact it
has entered the prothallium and made its way along the side
LIFE HISTORY OF PINUS 139
until it came into contact with the egg, when an entrance was
effected through the sheath-cells. That this has cost the pollen-
tube an unusual effort would seem to be evidenced by the fact
that it has become filled with a cytoplasm as dense as that of
the egg, whereas, normally, its cytoplasm is very scanty. If
it be true, as Lidforss ('99) claims, that the penetration of the
pollen-tube is simply due to a search for food, it would appear,
in such a case as this, that the pollen-tube is capable of very
intelligent searching. In this instance the relation of the pro-
thallium to the nucellar cap is very like that found in the more
primitive Gymnosperms such as Zamia, Cycas and Gtnkgo.
Here, however, the sperm-cells being non-motile it was neces-
sary, if fertilization take place at all, that the pollen-tube should
reach the egg.
The variations recorded here have a certain interest both
phylogenetically and ontogenetically ; but the most significant
lesson to be derived from them is the warning that they sound
against basing conclusions on meager observations. When
this is done, misconceptions and actual errors are bound to be
promulgated for truth.
BOTANICAL DEPARTMENT, WELLESLEY COLLEGE, Dec. 28, 1902.
NOTE.
This paper was completed on December 28, 1902. During
the time that has since elapsed much valuable literature dealing
with subjects more or less intimately connected with questions
herein discussed has appeared.
It is not feasible to make adequate references at this time to
all these papers, but the more important ones are mentioned be-
low, and the page in this paper, where mention of the views
recently expressed by other writers should have appeared, is
indicated.
The first 88 pages of this paper were printed before the writer
was able to obtain copies of some of the articles mentioned be-
low. I regret, therefore, that it was not possible in many in-
stances to refer by means of a foot-note to the references made
in this addendum.
Page 22. Strasburger ('04) now believes that synapsis is
OF THf
UN iv
Of
140 MARGARET C. FERGUSON
the most important stage in the heterotypic division and several
recent writers have expressed a similar opinion.
Page 26. The appearance of chromosomes from an " appar-
ently formless reticulum," described by Williams ('04) as occur-
ring in the first division of the tetraspore-mother-cell in Dictyota
is interesting in connection with the origin of the chromosomes
in the microspore-mother-cell in Pinus as herein described.
Page 31. Allen ('04) has described a somewhat similar
method of segmentation in the first division of the microspore-
mother-cell in Lilium Canadense.
Page 32. Strasburger ('04) has returned to his earlier view
regarding a true reducing or transverse division in the first
mitosis of the spore-mother-cell in plants.
Page 32. Farmer and Moore ('03), Williams ('04), Stras-
burger ('04) and others now accept the fact of a qualitative
division in plants.
Page 32. As a result of his recent study of Galtonia, Tra-
descantia, etc., Strasburger has decided that the forms of the
chromosomes which may occur in the anaphase of the hetero-
typic division are not the result of a double longitudinal spliting.
Page 33- According to Farmer and Moore ('03) the hetero-
typic division in both animals and plants is characterized by a
transverse division. This transverse division effects the sepa-
ration of the two chromosomes which constitute a bivalent
chromosome, and is therefore a qualitative or reducing division.
Page 34. This is in direct accord with the recent publica-
tions of Boveri ('04), Cannon ('03), Rosenberg ('03 and '04) and
others who have recently expressed themselves regarding the
individuality of the chromosomes.
Page 48. Strasburger's earlier observations on the pollen-
grain of Picea have now been confirmed by Miyake ('03) who
shows conclusively that the generative cell is cut off in Picea
before pollination takes place.
Page 62. In 1903 Miyake described and figured several
stages in the development of a single binucleated sperm-cell in
Picea.
Page 63. In a note at the close of Coker's ('03) paper on
Taxodium he says : " Miss Ferguson confirms Blackman's ('98)
LIFE HISTORY OF PINUS 141
statement that the sperm-cells of Pinus are furnished with a
cytoplasm of their own." But, as stated in 1901, I cannot
confirm Blackman's statement that each sperm-nucleus is sur-
rounded by its own cytoplasm.
Page 78. Strasburger ('04) states that in Taxus baccata a
heterotypical division occurs and that four megaspores are
formed which correspond to the four microspores formed within
the microspore-mother-cell.
Page 89. The nature and development of this tissue in
Taxodium, as described by Coker ('03), is essentially the same
as in Pinus. A preliminary note regarding the nature and
origin of the spongy tissue was published by the writer in 1903.
Page 97. Coker ('03) has made a similar observation in
Taxodium and Lawson ('04) finds that the nucleus of the ventral
canal-cell in Sequoia lies free in the cytoplasm of the egg.
Page 109. Both Wager ('04) and Williams ('04) have re-
cently expressed the view that the nucleolus contributes to the
bulk of the chromatin, either by storing or elaborating chromatin.
Page 124. The presence of two spiremes in the prophase of
the second division following fertilization and the conclusions
reached, as a result of this research, regarding the persistence
of the chromosomes are of especial interest in connection with
the discussions, appearing since the completion of this paper,
by Boveri ('04), Cannon ('03), Rosenberg ('03 and '04), and
others on the nature and individuality of the chromosomes.
Pages 127 and 132. Miyake ('03) has made a similar obser-
vation in Picea excelsa.
Page 129. Miyake ('03) finds that in Picea all three of the
nuclei, which pass into the egg from the pollen-tube but are
not directly concerned in fertilization, may divide before they
disintegrate. .
Page 136. Miyake ('03) has described conditions very simi-
lar in Picea and in Abies.
Page 137. Miss Opperman's ('04) paper has been published.
Page 137. As already stated, Coker ('02 and '03) finds this
to be the normal condition in Podocarpus and in Taxodium.
Lawson ('04) has described a similar condition in Sequoia, and
he finds that, normally, the nucleus of the ventral canal-cell
equals in size the egg-nucleus.
142 MARGARET C. FERGUSON
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LIFE HISTORY OF PINUS 145
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LIFE HISTORY OF PINUS 149
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LIFE HISTORY OF PINUS 1 53
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EXPLANATION OF FIGURES IN
PLATES I TO XXIV.
All figures were drawn with the aid of the Abbe* camera lucida. In some cases
a Zeiss microscope was used and in others a Bausch and Lomb. Various com-
binations of lenses were used with both instruments. The figures were reduced
one eighth in reproduction. The number accompanying the description of each
figure indicates the degree of magnification after the reduction.
Throughout the plates the lettering is to be interpreted as follows : prothal-
lium (pr.), first prothallial cell (_pr.i), second prothallial cell (/r.2), third
prothallial or antheridial cell (a.c.), tube-nucleus (/.».), stalk-cell (st.c.), stalk-
nucleus ($/.#.), generative cell (g*c. ), sperm-cell (s.c.), sperm-nucleus ($.#.)>
sperm-cytoplasm (s.cy.), spongy tissue (s./.), starch-grains (s-g"-), archegonium
(arch.), ventral canal-cell (v.c.), neck-cells («.c.), egg-nucleus (e.n.), cytoplasm
from the pollen-tube (c.p.t.), nutritive spheres (».s.), primary nucleolus (fiy.ns.),
secondary nucleolus (sy.ns.), receptive vacuole (r. v.~).
All the figures have been given their normal position, as nearly as it was
possible to do so, on the plates. That is, they are so placed that the primary
axis of the ovule would be parallel with the longer axis of the plates ; and the
portion of a figure nearest to the micropylar end of the ovule is always towards
the top of the plate.
( 154 )
PLATE I.
FIG. i. Two cells of the primitive archesporium showing the winter condition
of this tissue. X 1,400. Pinus austriaca. December 20, 1897.
2. A cell from the primitive archesporium in the early spring. Many of
the cells of the archesporium are undergoing division at this time.
X 1,400. Pinus austriaca. March 14, 1898.
3. A cell of the definitive archesporium, the microspore-mother cell, just
prior to the inception of its division. X 1,400. Pinus austriaca. April
27, 1898.
4. The same as fig. 3. X 1,400. Pinus Strobus. May 24, 1898.
5. The microspore-mother-cell approaching synapsis before a definite
spireme has been formed. X 1,400. Pinus austriaca. April 28, 1898.
6. The same as fig. 5. X 1,400. Pinus Strobus. May 24, 1898.
7. Synapsis. X 1,400. Pinus Strobus. May 24, 1898.
8. Recovery from synapsis, showing a continuous spireme. X 1,400.
Pinus Strobus, May 24, 1898. Material showing figs. 4 and 6 was col-
lected from a different tree than that showing figs. 7 and 8, and the
microspore-mother-cells were in a slightly different stage of division.
9. Complete recovery from synapsis. Chromatin in irregular granules,
on a broad linin band. X 1,400. Pinus Strobus.
10. The longitudinal splitting and transverse segmentation of the spireme.
Chromatin still distributed in irregular granules. X 1,400. Pinus
Strobus.
11. Longitudinal splitting completed, but the sister segments do not become
entirely disunited. Nucleoli still apparent. X I >4OO. Pinus Strobus.
12. a-e. Portion through the edge of a nucleus showing the twisting
of the" chromatic segments after longitudinal splitting. In most in-
stances these are not entire segments but portions that have been
severed by the microtome knife. The entire segments are very long
and coiled at this time. X 1,400. Pinus Strobus.
13. Early stage in the condensation and fusion of the longitudinally divided
spireme. Threads anastomosing in region of nucleoli. X 1,400.
Pinus Strobus.
(156)
PROC. WASH. ACAD. Set. VOL.
PLATE I.
M. C. F., DEL.
FERGUSON, -PINUS.
MICROSPOROGENESIS.
13
HELIOTYPE CO., BOSTON.
PLATE II.
FIG. 14. A more advanced stage in contraction, showing that adjacent threads
are anastomosing and fusing. X 1*400. Pinus Strobus.
15. A still more advanced stage in the fusion of the threads. Practically
all evidence of the earlier longitudinal fission has now disappeared.
X 1*400. Pinus Strobus.
16. The chromosomes becoming apparent. X 1*400. Pinus Strobus.
17. Distinct chromosomes, in the one half or reduced number, arising
from the contracted and more or less anastomosed skein. X 1,400.
Pinus Strobus.
18. a-c. Final stages in the formation of the chromosomes, showing the
separation of the segments from one another, and also the relation of
some of them to the nucleolus. X 1*400. Pinus Strobus.
19. a-l. Various forms of chromosomes observed before the organization of
the spindle. Each chromosome consists of two of the longitudinal
split segments which were formed immediately subsequent to synapsis.
X 1 5400. Pinus Strobus.
20. The chromatic segments completely differentiated. The remnant of a
nucleolus is still present, and the nuclear membrane is being resolved
into threads. X 1*400. Pinus Strobus.
21. An early stage in spindle-formation, showing kinoplasmic threads
entering from all directions but as yet no poles, or centers of radiations,
have been established. Chromosomes are homogeneous in structure
and regular in outline. X 1*400. Pinus Strobus.
22. The tripolar spindle. X 1,400. Pinus rigida. May 4, 1898.
23. The spindle has become nearly bipolar. X 1*400. Pinus rigida.
(158)
PROC. WASH. ACAD. Sci. VOL.
PLATE li.
M. C F., DEL.
HELIOTYPE CO., BOSTON.
FERGUSON.-PINUS.
MlCROSPOROGENESIS.
PLATE III.
FIG. 24. The equatorial plate stage. Spindle definitely bipolar and reaching to
the ectoplasm. X 1,400. Pinus rigida.
25, 26. The metaphase of the heterotypical division ; chromosomes irreg-
ular in outline and apparently much larger than in the late prophase.
X 1,400. Pinus Strobus.
27-29. Anaphase of the heterotypical division. The longitudinal split-
ting of the chromosomes has been very greatly delayed in some cases.
Such an appearance as that shown in fig. 29 is frequently met with,
the stretched arms of the daughter chromosomes extending nearly the
entire length of the spindle. X 1,400. Pinus Strobus.
30. The chromosomes just after reaching the poles, as seen in looking
down upon the end of the pole. X 1,400. Pinus Strobus.
31-34. Stages in the development of the daughter-nuclei. A definite
resting nucleus is formed at the close of the heterotypical division.
X 1,400. Pinus Strobus.
35. A late telophase in the first division, the daughter-nuclei fully estab-
lished. Delicate spindle threads still present, but no indication of a
cell plate. The wall of the microspore-mother-cell is beginning to
thicken centripetally. X 1,400. Pinus Strobus.
36-37. Stages in the formation of the spireme for the second division.
X J ,400. Pinus Strobus.
(160)
PROC. WASH. ACAD. Sci. VOL.
PLATE III.
35
M. C. F., DEL.
HELIOTYPE CO., BOSTON.
FERGUSON,-PINUS.
MICROSPOROGENESIS.
OF TH
UNIVE
OF
PLATE IV.
FIG. 38. Origin of the second spindle ; the chromatic band looped in region of
the future equatorial plate, and showing longitudinal fission. XM°°.
Pinus rigida.
39. Transverse segmentation is completed ; and the distinct chromosomes
have become apparent at the equatorial plate of the multipolar diarch
spindle. X 1,400. Pinus Strobus.
40. Separation of the daughter-chromosomes of each pair formed by the
transverse division shown in figure 39. X 1,400. Pinus Strobus.
41. Daughter-chromosomes arranged in two parallel rows at the equatorial
plate. XMOO. Pinus Strobus.
42. A late anaphase in the second division. X 1,400. Pinus Strobus.
43. Early telophase of the second division. X 1,400. Pinus Strobus.
44. Late telophase of the tetrad division ; the chromosomes of each nu-
cleus have fused to form a spireme, but the nuclear membrane is not
yet developed ; rather faint cytoplasmic threads connect the four nu-
clei ; the centripetal thickening of the mother-wall becomes more
apparent. X 1,400. Pinus rigida.
45. The tetrad division is completed and the young microspores are dis-
tinctly differentiated, each surrounded by its own wall. X1^00-
Pinus rigida. May 10, 1898.
46. The four microspores are separated by very prominent walls which
are continuous with the broad wall lining the original wall of the
microspore-mother-cell ; the outer, original spore-mother-wall is sepa-
rated at two points from the thick, more recently formed inner wall.
X 1,400. Pinus austriaca. May 9, 1898.
47. Microspores still within the mother-wall and showing the beginnings
of the wings or air-sacs. X 1,400. Pinus Strobus. May 30, 1898.
48. Rupture of the mother-wall and escape of the microspores. X Sio.
Pinus Strobus. May 30, 1898.
( 162)
PROG. WASH. ACAD. Sci. VOL.
PLATE IV.
FERGUSON, -PINUS.
MlCROSPOROGENESIS.
HELIOTYPE CO., BOSTON.
PLATE V.
FIG. 49. Empty wall of the microspore-mother-cell showing the compartments
formerly occupied by the microspores. X 810. Pinus Strobus.
50-54. Stages in the growth of the microspore ; the inner, partial wall
very apparent in the mature spore. Fig. 53 represents a section
through the middle of a young microspore in a plane perpendicular
to the wings. X 810. Pinus Strobus.
54-55. Stages in the first division of the microspore-cell ; the spindle
sharply pointed on the ventral side, broad on the dorsal side. X 810.
Pinus Strobus. June 7, 1898.
56. Telophase in the first division of the microspore. X 810. Pinus
Strobus.
57. The resting stage following the first division of the microspore.
X 810. Pinus Strobus.
58. The same as Fig. 57, but showing an exceptionally large prothal-
lial cell. X 810. Pinus Strobus.
59-60. Spireme-stage and early telophase in the division to cut off the
second prothallial cell. X&io. Pinus Strobus.
61. The germinated microspore at the close of the second division, show-
ing the first prothallial cell already in an advanced stage of disintegra-
tion. X 810. Pinus Strobus.
62-63. Stages in the third division of the microspore, showing the rapid
and almost complete obliteration of the first and second prothallial
cells. Both prothallial cells are cut off from the apical cell by definite
walls. X 8 10. Pinus Strobus.
(164)
PROC. WASH. ACAD. Sci. VOL.
PLATE v.
M. C. F., DEL.
FERGUSON.-PINUS.
DEVELOPMENT OF POLLEN-GRAIN.
HELIOTYPE CO., BOSTON.
OF THE
UNIVERSITY
OK
• IF
PLATE VI.
FIGS. 64-65. Mature pollen-grains ; in fig. 64 the remnants of the two prothal-
lial cells can be seen, while in fig. 65 all signs of the first cell have
disappeared. X^io. Pinus Strobus. June 9, 1898.
66. Vertical section through an ovule immediately after pollination ; the
macrospore-mother-cell is very conspicuous ; the upper portion of the
free limb of the integument is shown to be three cells in thickness,
there is a slight concavity in the apex of the nucellus ; macrospore-
mother-cell (m.m.c.), nucellar cap (#«c.), micropyle (mic.). X 46.
Pinus rigida. May 27, 1902.
67. Vertical section through the upper part of an ovule showing pollen-
chamber ; the middle layer of cells in the upper part of the free limb
of the integument has elongated and closed the microcarpylar canal.
X 46. Pinus rigida. June i, 1902.
68. A vertical section through the upper part of an ovule. The elongated
cells noted in fig. 67 have become divided by the formation of cross
walls into smaller cells. X 46. Pinus rigida. June 4, 1902.
69. A vertical section through an ovule some days after pollination. Axial
row (a.r.). X 62. Pinus Strobus. June 17, 1898.
70. A vertical section of an ovule showing the winter condition. X62.
Pinus Strobus. January 4, 1898.
71. A vertical section of an ovule soon after the second period of growth
has begun. X 62. Pinus Strobus. May 26, 1898.
72. A vertical section through the upper part of an ovule at the time of the
division of the generative nucleus ; (nuc.i), that portion of the nucellar
cap which was developed during the first period of activity; (nuc.2),
that portion of the nucellar cap which constitutes the second year's
growth; o, disintegrating spongy tissue. X62. Pinus Strobus.
June 9, 1898.
(166)
PROC. WASH. ACAD. Sci. VOL.
PLATE VI.
o - _-.._
7 I
M. C. F., DEL.
HELIOTYPE CO., BOSTON,
FERGUSON,-PINUS.
POLLINATION AND SUBSEQUENT PHENOMENA.
PLATE VII.
FIG. 73. A vertical section through the upper part of an ovule shortly before fer-
tilization ; reconstructed from three adjacent sections of the series ; 0,
last vestige of spongy tissue. X 62. Pinus Strobus, June 15, 1898.
74. Pollen-grain from the nucellus of Fig. 73. The antheridial cell is still
undivided. X472.
75. A vertical section through the extreme upper portion of an ovule soon
after pollination, showing the uppermost part of the nucellar cap, and a
pollen-grain in the first stages of germination ; p.c, pollen-chamber.
X472. Pinus Strobus. June 13, 1898.
76. A pollen-grain soon after germination. The tube-nucleus is moving
into the pollen-tube. X 472. Pinus Strobus. June 24, 1898.
77. A pollen-grain after the tube-nucleus has passed into the pollen-tube.
X472. Pinus Strobus. July 15, 1898.
78. Spireme stage in the division of the antheridial cell. X I >4QO.
Pinus rigida. April 27, 1898.
79-80. Stages in the division of the antheridial cell. Xi>4°°. Pinus
Strobus. August 4, 1898.
81. A pollen-grain after the antheridial cell has divided. X 472- Pinus
Strobus. August 4, 1898.
82. The same at a later date, showing a slight increase in the size of the
generative cell. X472. Pinus Strobus. October 7, 1898.
(168)
PROC. WASH. ACAD. Sci. VOL.
PLATE VII.
C. F^f DEL.
HELIOTYPE CO., BOSTON.
FERGUSON, -PINUS.
GROWTH OF THE POLLEN-TUBE.
Proc. Wash. Acad. Sci., September, 1904.
PLATE VIII.
FIG. 83. The pollen-tube which is shown in fig. 70, more highly magnified.
X 472. Pinus Strobus. January 4, 1899.
84. A pollen-grain and the upper portion of a pollen-tube, showing the
stalk- and the generative-cell just before their passage into the pollen-
tube. X 472« Pinus austriaca. May 3, 1898.
85, 86. Later stages than the above, showing the passage of the generative-
and the stalk-cell into the pollen-tube ; in fig. 86, the two cells are
breaking loose from each other. X472- Pinus austriaca. May 10
and 17, 1898.
87. The male gametophyte at the time of the entrance into the tube of the
generative- and the stalk-cell ; n.t, a bit of the dead nucellar tissue.
X472- Pinus Strobus. June 9, 1898.
88. A pollen-grain after the generative and the stalk-cell have passed into
the pollen-tube ; taken from the top of the nucellus of the ovule shown
in fig. 72. X472- Pinus Strobus. June 9, 1898.
89. A few of the cells from that portion of the nucellar cap marked nuc.2
in fig. 72. The cells are filled with starch grains. X 472. Pinus
Strobus. June 9, 1898.
90-92. Portions of pollen-tubes showing successive stages in the passage
of the stalk-cell over the generative cell, as also the presence of large
quantities of starch in the pollen-tube. X472- Pinus resinosa. June 2,
P. Strobus, May 24 ; P. rigida, June 8, 1898.
93. The generative cell, bearing on its surface both the tube-nucleus and
the stalk-nucleus. In this instance the stalk-cell has passed beyond
the tube-nucleus. X 472- Pinus resinosa. June 3, 1898.
94. The generative cell showing a very early stage in the formation of the
spindle. The nucleus is in the extreme uppermost part of the cell.
X 744- Pinus rigida. June 8.
(170)
PROC. WASH. ACAD. Sci. VOL.
PLATE VIII.
st.c
94
M. C. F., DEL.
HEUOTYPE CO., BOSTON.
FERGUSON, -PINUS.
SPERMATOGENESIS.
PLATE IX.
FIGS. 95-96. The generative cell in the early stages of its division, showing
granular condensation and radial arrangement of cytoplasm. The
spindle fibers arise in the cytoplasmic condensation and extend in the
form of a cone to the nuclear membrane. X 744- Pinus rigida.
June 8 and 10, 1898.
97. A cross-section through the generative cell during an early stage in
its mitosis. The protoplasmic condensation is seen from below
looking toward the nucleus. X 744- Pinus austriaca. June 4, 1898.
98. A later stage in the division of the generative nucleus. X 744- Pinus
austriaca, June 10, 1898.
99. The generative cell just before the disappearance of the lower portion
of the nuclear membrane showing a single deep indentation on the
lower side of the nucleus. X 744- Pinus Strobus. June 9, 1898.
100. A stage in spindle-formation directly following that shown in fig. 99.
The nuclear membrane has given way and the spindle fibers are enter-
ing the nuclear cavity. The nucleolus is still distinctly visible.
X 744- Pinus Strobus. June 10, 1898.
101. The gradual disappearance of the nuclear membrane and the extension
of the spindle fibers across the nucleus. X 744- Pinus austriaca.
June 7, 1898.
102-103. Further development of the spindle and the formation of the
chromosomes. The marked condensation in the cytoplasm from
which the spindle arose has almost entirely disappeared. X 744-
Pinus austriaca. June 8, 1898.
(172)
PROC. WASH. ACAD. Set. VOL.
PLATE IX.
101
«. C. F., DEL.
102
FERGUSON, -PINUS.
SPERMATOGENESIS.
HEUOTYPE CO., BOSTON.
PLATE X.
FIGS. 104-106. Later stages in the development of the spindle showing the gradual
drawing together of the outer extremities of the threads to form the
upper pole of the spindle. The upper pole of the spindle does not
reach the nuclear membrane, but in fig. 105 definite threads extend
from the pole to the nuclear membrane. X 744- Fig. 104. Pinus
rigida, June 13 ; the other figures, Pinus austrtaca, June 9-10, 1898.
107. First stage in the development of the sperm-nuclei. X 744- Pinus
Strobus. June 9, 1898.
108. The sperm-nuclei just after the formation of the nuclear membrane
showing early stages in the development of the daughter-reticula.
The lower nucleus is already slightly larger than the upper one.
X 744- Pinus montana uncinata. May 31, 1898.
109-112. Various stages in the growth of the sperm-nuclei. A cell plate is
sometimes apparent as in fig. no, but no dividing wall is ever formed.
X 744- Fig. 112. Pinus Strobus, June 10; fig. 109, P. resinosa, June
15; fig. no, P. austriaca, June 10. Fig. in represents another sec-
tion through the upper nucleus of fig. 1 10, and shows how the- upper
of the sperm-nuclei is frequently indented along its outer surface.
1898.
(174)
PROC. WASH. ACAD. Sci. VOL.
PLATE X.
m
\JSfm
104
105
106
M. C. F., DEL
FERGUSON, -PINUS.
SPERMATOGENESIS.
PLATE XI.
FIG. 113. A peculiar figure sometimes observed in the late telophase of the
division. X 744- Pinus austriaca. June 10, 1898.
114. A pollen-tube in which the smaller sperm-nucleus appears to be in ad-
vance of the larger. This pollen-tube, having approached an egg
that had already been fertilized, has turned aside and is passing up
over the endosperm so that the normal position of the cells appears
exactly reversed; n.c., neck-cells of the archegonium. X 289- Pinus
Strobus. June 20, 1898.
115-116. Cross-sections through the two sperm-nuclei after they have
attained full size and have about reached, in their downward passage,
the middle of the nucellar cap. X 744- Pinus Strobus. June 15, 1898.
117. The sperm-cell after all traces of the spindle have disappeared, but
before the two nuclei have come together. X 472. Pinus Strobus.
June 13, 1898.
118. The same after both nuclei have come to lie in the upper part of the
cell. X 472. Pinus Strobus. June 10, 1898.
(176)
PROC. WASH. ACAD. Sci. VOL.
PLATE XI.
^BtJWSKyaeY?^
:^MbS>^>^.
--
«t.c.
M C. F., DEL.
HELIOTYPE CO., BOSTON.
FERGUSON,-PINUS.
SPERMATOGENESIS.
PLATE XII.
FIG. 119. The lower portion of a pollen-tube which has penetrated about two-
thirds the length of the nucellar cap. X 472. Pinus Strobus. June
14, 1898.
1 20. The lower portion of a pollen-tube which is just pushing between
the neck-cells of the archegonium. f, pit in apex of tube. X 472-
Pinus Strobus. June 20, 1898.
121. A vertical section of a young cone; the ovuliferous scales have not as
vet been organized. X 57- Pinus austriaca. March 14, 1898.
122. Section of an ovuliferous scale showing the first indication of an
ovule, m. ovule ; o.s, ovuliferous scale ; b, bract. X 150* Pinus
Strobus. May 3 1, 1898.
123. A vertical section of an ovule one week later than that shown in
fig. 122. X I5°- Pinus Strobus. June 6, 1898.
124. A very young macrospore-mother-cell showing differentiation of
spongy tissue. X 394- Pinus rigida. May 15, 1902.
125. The macrospore-mother-cell from fig. 124 more highly magnified.
X8io.
126. A macrospore-mother-cell just prior to synapsis. X 810. Pinus
Strobus. June 27, 1898.
(178)
PROC. WASH. ACAD. Set. VOL.
PLATE XII.
4
:^':cT •• ::$8&v~^-'
\t ° O O _'§S ^0
124
M. C. F., DEL.
FERGUSON, -PINUS.
MACROSPOROGENESIS.
126
HELIOTYPE CO., BOSTON.
OF THE \
^NIVERS/TY )
OF
PLATE XIII.
FIG. 127. The macrospore-mother-cell in synapsis. X 810. Pinus austriaca.
June 6, 1898.
128. The same in recovery from synapsis showing continuous skein.
X 810. Pinus austriaca.
129-133. Stages leading to the organization of the chromosomes in the
first or heterotypical division of the macrospore-mother-cell. X 810.
Fig. 132, Pinus Strobus, the others, P. rigida. Fig. 131 illustrates an
instance in the unusually early disappearance of the nuclear membrane.
I34~I37- Stages in the establishment of the spindle in the first division of
the macrospore-mother-cell. The reduced or one half number of
chromosomes appear in this mitosis. The spindle arises as a multi-
polar diarch. X 810. Fig. 137, Pinus rigida, the others, P. Strobus.
138. Late telophase in the first division. A cell-wall is laid down and defi-
nite resting nuclei are formed. X 810. Pinus Strobus. June 13, 1899.
139-140. The close of the heterotypical division. Resting nuclei are
formed but the upper resting nucleus in each case shows signs of
disintegration and doubtless would not have divided. X 810. Fig.
139, Pinus austriaca, fig. 140, P. rigida.
141. The two daughter-cells formed by the first division of the macrospore-
mother-cell. Both would doubtless have divided again. X 810.
Pinus austriaca.
142. The second or homotypic division of the macrospore-mother-cell.
The spindles are oblique and arise as multipolar diarchs. The chro-
mosomes have the same form as those which arose on the first division
of the macrospore-mother-cell. X 810. Pinus austriaca.
(180)
PROC. WASH. ACAD. Sci. VOL.
PLATE XIII.
M. C. F., DEL.
141
FERGUSON.-PINUS.
MACROSPOROGENESIS.
HELIOTYPE CO., BOSTON.
"
OF THE
ITY
PLATE XIV.
FIGS. 143-144. Two axial rows of three cells each. The upper of the two daugh-
ter-cells formed as a result of the heterotypical division has not divided
in either case ; a few starch grains in the cells of the axial row and
many large ones in the spongy tissue as shown in fig. 143. X 810.
Fig. 143. Pinus Strobus y fig. 144, P. rigida.
145. An axial row of four cells, reconstructed from serial sections. X 810.
Pinus austriaca.
146. A macrospore nucleus surrounded by large starch grains. X^io.
Pinus austriaca.
147. Growth of the functional macrospore ; the peripheral layer of cyto-
plasm already established ; the three upper cells of the axial row almost
destroyed ; one large cell of the spongy tissue shown. X 810. Pinus
austriaca. June 13, 1898.
148. An axial row of three cells ; the functional macrospore much enlarged,
and the two upper cells in an advanced stage of disintegration ; the
spongy tissue distinctly differentiated ; the cells along its outer sur-
face more or less tabular in outline and many of them badly disorgan-
ized. Pathological conditions have just entered in as shown by the
reduced amount of cytoplasm in the cells of the spongy tissue and
the slight thickening of their walls. X 234- P* rigida. June 24,
1902.
149. The first division of the macrospore-nucleus. X234- Pinus Strobus.
July 29, 1898.
150. The karyokinetic figure from the above more highly magnified ; the
division conforms to the typic type and shows the one-half number of
chromosomes. X 810.
151. The first two nuclei of the female gametophyte. X234- Pinus aus-
triaca. July 29, 1898.
152. The four-nucleated stage of the female gametophyte. X234- Pinus
Strobus. August 4, 1808.
153. One of the sixteen free nuclei of a female gametophyte, all sixteen
nuclei being in the spireme stage of division. X 810. Pinus Strobus.
October 12, 1898.
154. A vertical section of the central portion of an ovule showing .the
spongy tissue and the prothallium with its nuclei, of which there are
sixteen, all in the equatorial stage of division ; the prothallium has
been somewhat displaced by the action of the fixing fluid. X 46.
Pinus Strobus. October 12, 1898.
155. One of the spindles from the above more highly magnified. X 744-
(182)
PROC. WASH. ACAD. Sci. VOL.
PLATE XIV.
154
M. C. F., DEL.
FERGUSON, -PINUS.
GERMINATION OF MACROSPORE.
HELIOTYPE CO., BOSTON.
PLATE XV.
FIG. 156. Surface view of a bit of the prothallium showing two free nuclei and
the vacuolate protoplasm surrounding them. X 744- Pinus Strobus.
May 17, 1898.
157. A radial section through the lower portion of an ovule showing pro-
thallium, spongy tissue, and normal nucellar tissue. X472« Pinus
Strobus. May 26, 1899.
158. As fig. 157, except that the spongy tissue and the normal nucellar
tissue are separated by a double layer of cells, belonging to the nu-
cellus, which have lost their protoplasmic content but their walls have
not yet collapsed. X 472- Pinus Strobus, May 26, 1899.
159. A bit of the prothallium in surface view showing the complex cytoplas-
mic figure characteristic of the late telophase in free nuclear division.
X472. Pinus austriaca. May 17, 1898.
160. Surface view of a portion of a prothallium immediately after the or-
ganization of cell-walls separating the free nuclei. X 472- Pinus
Strobus. May 26, 1899.
161. A bit of the prothallium as seen in radial section just after cell-walls
have arisen. The cells are open on their inner surfaces and the
nuclei remain near the open sides. X 394- Pinus austriaca. May
20, 1898.
162. A prothallium still open at the center showing that true "alveoli" as
described by Sokolowa are not present ; the archegonia rudiments at
the micropylar end ; the spongy tissues still prominent. X 62. Pinus
austriaca. May 24, 1898.
163. A condition often found in the ovule. The macrospore-mother-cell
has failed to develop and the walls of the spongy tissue have thickened
and stain deeply. X 46. Pinus Strobus.
164-166. Figures illustrating karyokinesis in the spongy tissue. The
method is typic with the number of chromosomes characteristic of the
sporophyte. X 810. Pinus Strobus.
(184)
PROC. WASH. ACAD. Sci. VOL.
PLATE XV.
r-T'
r
"
HELIOTYPE CO., BOSTON.
FERGUSON,-PINUS.
FEMALE PROTHALLIUM.
Proc. Wash. Acad. Sci., September, 1904.
PLATE XVI.
Pinus Strobus unless otherwise indicated.
FIG. 167. Telophase in the division of a cell of the spongy tissue. X 810.
168. The macrospore and some of the cells of the spongy tissue in the
first stages of disintegration and having the appearance of a group of
sporogenous cells, (mac.) macrospore. X<X>. Pinus austriaca.
169-175. Stages in the early development of the archegonium. The
central cell remains close beneath the neck cells. The cytoplasm is
very vacuolate. X I4°-
Fig. 169, May 26, 1890; fig. 171, May 31, 1898.
176-179. Later stages in the growth of the archegonium. The vacuoles
gradually disappear and many proteid vacuoles arise in the cytoplasm.
X 62. Fig. 178 collected June 15, 1899.
180. Mature archegonium. The nucleus has assumed a central position in
the cell ; the ventral canal-cell is in an advanced stage of disintegration ;
the proteid vacuoles are distributed about the periphery especially
along the basal portion of the egg, the receptive vacuole has appeared
but has not yet assumed its mature or final shape. X 62. June 17,
1899.
181. Nucleus of the central cell shortly before its division. This nucleus
is almost invariably concave on the side towards the neck cells. X472'
182-184. Prophases in the division of the central cell. X 472- Fig. 184,
Pinus austriaca.
(186)
PROC. WASH. ACAD. Set. VOL.
PLATE XVI.
67
«^0"
W rfr
^Ssfr
168 W"
pr.
176
!
177
178
70
173
174
179
182 via
~£*^'-- '• '
^-« i't ^V?^7"
181
M. C. F., DEL.
FERGUSON ,-PINUS.
OOGENESIS.
HELIOTYPE CO., BOSTON.
OF THE
UNIVERSITY
OF
PLATE XVII.
Pinus Strobus unless otherwise indicated.
FIGS. 185-186. Later stages in the prophase of the division of the central cell.
X472. Fig. 186, Pinus austriaca.
187-188. Disappearance of the nuclear membrane and establishment of the
achromatic spindle. The spindle now lies wholly within the area pre-
viously occupied by the nucleus. X 472.
189. Cross-section of the nucleus of the central cell just as the chromo-
somes are undergoing longitudinal splitting at the equatorial plate.
X472.
190-197. Separation of the half chromosomes and formation of the daughter-
nuclei. X 472. Figs. 192 and 195, Pinus austriaca. These figures
show some of the variations occurring in the mitotic figure for this
division, and the corresponding variations in the structure of the
nucleus of the ventral canal-cell. Figs. 190, 191, 193 and 196 are very
interesting, showing how some at least of those ventral canal-cells in
which no definite nucleus is organized have arisen. Figs. 192, 194
and 195 are also interesting as leading to the formation of a normal
nucleus within the ventral canal-cell. It will be noted that this spindle
is always monopolar at its lower extremity and usually broadly multi-
polar at the opposite end. Fig. 192 is the only instance observed of a
sharply bipolar spindle. The egg nucleus is larger from the very
first than the nucleus of the ventral canal-cell.
(188)
PROC. WASH. ACAD. Sci. VOL.
PLATE XVII.
7i
195
M. C. F., DEL.
197
HELIOTYPE CO., BOSTON.
FERGUSON,— PINUS.
OOGENESiS.
PLATE XVIII.
Pinus Strobus.
FIGS. 198-199. Some of the aspects presented by the ventral canal-cell. It is
doubtful in both of these cases if any nucleus has ever been organized
within the ventral canal-cell, and the chromosomes have not even
fused to form a spireme. X 472-
200-202. Later history of the ventral canal-cell and early stages in the
development of the egg-nucleus. The first indication of the primary
nucleolus is seen on the lower side of the egg-nucleus in fig. 202, and
the ventral canal-cell already shows marked signs of disintegration.
X472-
203-204. Later stages in the downward movement and growth of the egg-
nucleus showing growth of primary nucleolus. X 472.
205. Mature egg-nucleus. The primary nucleolus is very large and vacuo-
late and several secondary nucleoli are scattered throughout the nu-
cleus. The structure of this nucleus varies greatly. This one was
selected not because it can be said to be any more typical than others,
but because it represents an average rather than an extreme condition
as to density of reticulum and number of secondary nucleoli. X 472«
(190)
PROC. WASH. ACAD. Sci. VOL.
PLATE XVIII.
199
200
s>- i -W
201
f> •%'><•-.
itPiltfii
\#
M. C. F., DEL.
205
FERGUSON,-PINUS.
OOGENESIS.
R^««WfSl3»
-fttiferf* jrx*-bPV% fe
HELIOTYPE CO., BOSTON.
PLATE XIX.
Pinus Strobus unless otherwise indicated.
FIG. 206, a-g. Portions of the reticulum from different mature egg-nuclei,
showing some of the variations which may occur in the structure of
this nucleus. X 1050.
207. Division of the central cell, showing also the lower portion of a pollen-
tube which has already reached the endosperm. In this instance a
very short time would have elapsed between the division of the central
cell and fertilization. X 2°9« Pinus montana uncinata.
208. The primary nucleolus from a mature egg-nucleus with secondary
nucleoli clustered about it and evidently formed by it. The primary
nucleolus has a great affinity for stains at this time. X IO5°-
209. The primary nucleolus of a mature egg-nucleus. This nucleolus
shows a weak reaction towards dyes, and apparently has an outer,
limiting membrane. X IO5°«
210. The framework of a primary nucleolus from a mature egg-nucleus.
This nucleolus has remained of a light greenish-yellow color after
treatment with Flemming's triple stain. X IO5°-
211. The upper part of an archegonium showing cavity, the receptive
vacuole, formed in the cytoplasm just prior to fertilization. X I4°-
212. The upper part of an archegonium just after the entrance into the egg
of the elements from the pollen-tube. X 140.
213. A slightly later stage. The cytoplasm of the sperm-cell has already
fused with the cytoplasm of the egg. X I4°-
214. An entire archegonium showing the sexual nuclei in contact, and,
above them, the various elements which have come into the egg from
the pollen-tube. X 62. June 21, 1898.
215. The upper part of an archegonium in the same stage as the above.
X HO-
(192)
PROC. WASH. ACAD. Sci. VOL.
J-
PLATE XIX.
206a
206g
206e
V
206b
206f
212
*<^H^ 2I4
FERGUSON, -PINUS.
FERTILIZATION.
207
209
208
I
210
' - : •-•' •:--x%e-. •'••.;>
'^fciW^'il
215 S£8SRg&
-"' 'Vt*'-
HELIOTYPE CO., BOSTON.
PLATE XX.
Pinus Strobus.
FIG. 216. The sexual nuclei just before coming into contact. Note depression.
in egg-nucleus. X 140- June 17, 1899.
217-223, a. Various appearances presented by the conjugating nuclei. It
will be borne in mind that these figures are so placed that the major
axis of the archegonia in which they occur would be parallel with the
longer axis of the plate. As a rule the sexual nuclei differ structurally
in size only. X J4°-
223. b. Another section through the egg-nucleus shown in fig. 223, a. There
is a greater difference in the size of the conjugating nuclei than would
appear in fig. 223, a, which is cut obliquely through the egg-nucleus.
X 140-
224. An early prophase in the first division following fecundation. Show-
ing early separation of chromatic from achromatic substance. X 472-
225. A slightly later stage. The cytoplasm caught between the two nuclei
has collected into spherical masses. X 472-
226. A still later stage in the formation of the two chromatic spiremes.
X472-
227. A still later stage in which the paternal chromatic spireme has taken
up a position near the maternal spireme, and a few delicate achromatic
threads have made their appearance in the neighborhood of these
spiremes. The nuclear membranes are still present, but have broken
down at several points. X 472>
228. A later stage. The nuclear membrane has entirely disappeared ; the
spindle fibers have increased in number ; and the rearrangement of
the achromatic, nuclear reticula into granular threads is very apparent.
X472.
229. More advanced stage in the formation of the spindle. The spindle is
distinctly multipolar in origin. X 472-
(194)
PROC. WASH. ACAD. Sci. VOL.
PLATE XX,
• '
fei
226
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•
FERGUSON,-PINUS.
FERTILIZATION.
HELIOTYPE CO., BOSTON.
THE
OF
PLATE XXI.
Pinus Strobus.
FIG. 230. The spindle fibers have become more abundant and transverse segmen-
tation of the spiremes has occurred at some points. X 472-
231. The spindle fully established having now assumed the form of a
multipolar diarch ; the two chromatic spiremes still perfectly distinct.
X472.
232. The two spiremes after segmentation; the two halves of the spindle
seem to indicate the maternal and the paternal portions of the mitotic
figure. X 472.
233. Early stage in the formation of the chromosomes. The chromatic
elements still occur in two distinct groups, but position, alone, deter-
mines which are maternal and which are paternal. The segments can
not be structurally differentiated. X 472.
234. The chromosomes being oriented at the nuclear plate. The distinc-
tion between paternal and maternal elements no longer evident.
X472.
235. A cross-section through the nuclear plate just before the separation
of the chromosomes ; twenty -four segments are distinctly shown.
X472.
236-238. Some of the aspects presented by this mitotic figure during meta-
kinesis. X472.
239. An anaphase of the mitosis. X472-
240. A late anaphase of the division ; the poles terminate in granular areas
from which delicate threads extend into the cytoplasm ; some of the
nucleolar substance from the egg-nucleus still persists. X472.
241. One end of the spindle in the same stage as the above ; the fibers which
radiate from the polar region of the spindle are very abundant and
stain deeply. X 472.
(196)
PROC. WASH. ACAD. Sci. VOL.
PLATE XXI
™
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•.'•-_ ; 7%|&
237
230
232
231
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FERGUSON,-PINUS.
FERTILIZATION.
234
236 '
240
•
HELIOTYPE CO.. BOSTON.
A .
OF THE
UNIVERSITY
OF
PLATE XXII.
Pinus Strobus.
FIG. 242. One aspect presented by the karyokinetic figure in the telophase of
this division. X 472-
243. The two segmentation-nuclei fully formed. X472«
244. One of the two segmentation-nuclei in an early prophase of divi-
sion. X 472.
245-246^. Later stages in the second division, showing two chromatic
spiremes. X472.
247. A still later stage. The two groups of chromosomes can still be made
out. X472.
248. An entire archegonium showing the position of the two segmenta-
tion-nuclei during division. The receptive vacuole has been distorted
by the entrance of the contents of the pollen-tube. X 62.
249. An archegonium showing the original position of the four segmenta-
tion-nuclei. X^2.
2500:. The same after the nuclei have begun their downward movement.
X62.
250^. A nucleus from 250** showing details of its structure and fibers in
the surrounding cytoplasm. X 472«
25 la. An archegonium after the nuclei have almost reached the base of the
oosphere. X^2.
251^. A portion of fig. 25 la, showing details in nuclear structure, and
fibers in the surrounding cytoplasm. X472-
(198)
PROC. WASH. ACAD. Sci. VOL.
PLATE XXII.
•— &~
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243
245
244
246b
247
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». C- F., DEL.
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FERGUSON,— PINUS.
DEVELOPMENT OF PROEMBRYO.
25lb
HELIOTYPE CO., BOSTON.
UNIVERSITY
OF
PLATE XXIII.
Pinus Strobus unless otherwise indicated.
FIG. 252^. The lower part of an archegonium after the four nuclei have ar-
ranged themselves at the " organic apex " of the oosphere. X^2.
252^. A portion of the above ; the nucleus is in the early prophase of
division ; the cytoplasm surrounding the nucleus has became dense
and deeply staining. X472.
253^. The basal portion of an egg ; the four segmentation-nuclei are in the
metaphase of the mitosis. X^2. June 19, 1899.
253^. A part of the same showing details. X 472-
254*7. A portion of a lower part of an oosphere after the formation of the
eight nuclei of the proembryo. X 62.
254^. A part of the above giving details. No cell-walls have as yet been
formed, but there is a slight differentiation of the cytoplasm about each
nucleus. X 472.
255*7. A somewhat later stage than fig. 254*7. X 62.
255$. An enlarged portion of the above, showing cell-walls in the process of
formation. X 472.
256. Vertical section through the base of an archegonium showing that the
four nuclei of the upper tier of cells in the proembryo divide before
any divisions occur in the four lower cells. X 96. Pinus austriaca.
257-258. Figures occurring in the upper part of archegonia during the
division of the segmentation-nuclei. These doubtless represent the
smaller sperm-nucleus. X472-
259<z-259#. Figures occurring in the upper part of an archegonium at the
time of the second division following fertilization ; fig. 259*7 represents
the tube-nucleus ; the karyokinetic structure in fig. 259^, is the smaller
sperm-nucleus, and just above it the stalk-cell is still distinctly visible.
X472.
260. Two macrospore-mother-cells. X 830. Pinus rigida. June 7, 1902.
261. An axial row showing oblique wall between two of the spores. X 394-
Pinus austriaca. June 13, 1898.
262. a. A section through a prothallium showing unusual origin of
archegonia from cells several layers deep in the prothallium. X 75
(200)
PLATE XXIII.
PROC. WASH. ACAD. Set. VOL
160
M. C F., DEL.
HEUIOTYPE CO., BOSTON.
FERGUSON,-PINUS.
DEVELOPMENT OF PROEMBRYO.
Proc. Wash. Acad. Sci., Sep., 1904.
PLATE XXIV.
FIG. 262*. Another section through the same prothallium as that shown in 262^.
Altogether there are more than twenty archegonia formed in the upper
part of this prothallium. X 75-
263. Archegonia formed not only at the top but along the sides of the en-
dosperm. Reconstructed from several sections. Seven of the arche-
gonia are visible in a single section. X 31- Pinus montana uncinata.
June 10, 1898.
264. Linear arrangement of archegonia through the center of the prothal-
lium. All of these archegonia are connected with the exterior by a
passage above the neck-cells, which does not show in this view, but in
the lower ones it leads to the side of the prothallium, rather than to
the top. X31- Pinus austriaca, June 17,1898.
265. A little archegonium " budding" from a sheath cell of a larger arche-
gonium. X 53- Pinus resinosa. June 15, 1898.
266. A smaller archegonium at the base of a larger one and opening into
it. The smaller one has no neck-cells and the nucleus of its cen-
tral cell has evidently been derived from one of the sheath-cells of the
upper archegonium. X3*- Pinus rigida. June 13, 1898.
267. The same as fig. 266 except that the central cell of the lower archego-
nium has divided and the egg has reached maturity, while the nucleus
of the central cell of the smaller upper archegonium has not divided.
X 46. Pinus resinosa. June 24, 1898.
268. The largest ventral canal-nucleus observed in Pinus Strobus. There is
no wall present cutting off a ventral canal-cell, but the nucleus is free
in the cytoplasm of the egg. X 3*- June 14, 1899.
269. An archegonium showing the only nucleus of such a large size ob-
served in any species for the ventral canal-nucleus. X 46- Pinus
austriaca. June 2, 1898.
270. Fragmentation of the egg-nucleus. X 46. Pinus Strobus. June 15,
1899.
271. A pollen-grain after germination showing an increase in the normal
number of nuclei. X472- Pinus austriaca. May 17, 1898.
272. The generative cell and another nucleus, not the stalk-nucleus, just
passing into the pollen-tube. X 472- Pinus Strobus. May 20, 1898.
273. The generative cell and another cell passing into the pollen-tube and
followed by the stalk-cell. Presumably two generative cells have been
formed. X 394- Pinus rigida. May 3, 1898.
274. An archegonium after fertilization. One of the two segmentation-nu-
clei has divided while the other has not. X 46- Pinus Strobus.
275. An instance in which the greater portion of the upper end of the pro-
thallium is separated by a considerable space from the nucellar cap. A
pollen-tube not able to cross this space and enter between the neck
cells has effected entrance into the side of an archegonium, and the
four segmentation-nuclei have been formed ; the fifth nucleus is evi-
dently the smaller sperm-nucleus ; the very small nucleus at the
top may be the ventral canal-nucleus, but more probably it is the tube-
nucleus. X31- Pinus Strobus. June 15, 1899.
(202)
PROC. WASH. ACAD. Sci. VOL.
PLATE XXIV.
262b
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264
271
265
266
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268
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M. C. F., DEL.
270
269
272
HELIOTYPE CO., BOSTON.
FERGUSON,-PINUS.
ABNORMALITIES.
14 DAY *,
R. ,.AN TO DESK FROM Wi. x , b >R .O 3 D
This book is due onthe last date stamped below, or
on the date to which renewed.
Renewed books are subject to immediate recall.
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